Biomedical Journal of Scientific & Technical Research (BJSTR) is a multidisciplinary, scholarly Open Access publisher focused on Genetic, Biomedical and Remedial missions in relation with Technical Knowledge as well.
Written by Arsi, Rizal Andi Syabana, Supyani Dwiwiyati Nurul Septariani, Ryan Budi Setiawan Tita Widjayanti, Tili Karenina & Junairiah, this book contains eight chapters. Chapter 1 is about the history of the discovery and importance of viruses, Chapter 2 regarding the nomenclature of plant viruses, Chapter 3 regarding symptom recognition, Chapter 4 regarding transmission and spread of viruses, Chapter 5 regarding control of plant diseases caused by viruses, Chapter 6 regarding the history of the discovery and importance of viruses, Chapter 7 regarding virus ecology and epidemiology, Chapter 8 is about the basics of plant virus diagnosis. Hopefully this virology book can increase readers’ insight into the viruses that are currently attacking. Technological developments can contribute to identification of virus development over time. The authors really hope for input and criticism to improve the book. Constructive input and criticism that improves this book is very useful for the authors to write better in the future. Viruses are very small living creatures that can develop and grow in living cells. The virus cannot reproduce itself without any living creatures to host. Viruses don’t have cellular equipment in multiplying the virus. Viruses have a protective device in the form of a protein layer or known as a capsid. The protective layer found on viruses encloses nucleic acid molecules and DNA or RNA only. Viruses have a network which is a network that can convey genetic information to the virus. Viruses can also reproduce or replicate on the living creatures they host. Viruses can reproduce themselves if they are inside living things, but they will form crystals when it is in dead cells or inanimate objects. Viruses are microorganisms that are very small in size. To get an idea regarding viruses, tools such as an electron microscope are needed. The virus cannot be seen directly, but the symptoms that caused by viruses can be seen in infected plants. Plants attacked by viruses can cause characteristic symptoms.
This is due to changes that occur in the plant. Viruses can be seen using a light microscope. Viruses in plants can be seen directly by the symptoms they cause by viruses. Plants that are attacked by viruses can be caused by the presence of insect vectors that transmit disease to the plant. The vector insects that cause symptoms of viral diseases in plants have piercing sucking mouthparts or haustelata. However, the virus attacked plants depend on the presence of wounds or insect vectors that help in infection process in the plant. Viruses that attack plants can cause major losses because viruses can cause failure of production in plants. There are so many viruses that can cause disease. Viruses that cause disease in plants are found in many food crops and horticultural crops. Viruses that attack via insect vectors have similar symptoms caused differently depending on the insect that transmits the disease to the plant. Viruses have the ability to reproduce themselves. However viruses do not have structures like living creatures (Aji, et al. [1-5]. (Yuliani, et al. 2006) Viruses have different meanings, this is due to influence identification in technology and biomolecular in carrying out virus identification. Apart from that, the chemical and physical properties of the virus can be influenced. A virus can be interpreted as a pathogen that can cause disease on plants which can be transmitted through scratches and insect vectors. Viruses in their reproduction use ribosomes and the host plant cells they host, this is because viruses are pathogens that are obligate parasites. Where viruses will not develop or replicate without a host as a place life.
So an obligate parasite is a type of parasite that cannot live in living cells, if it is in a dead cell then the parasite cannot replicate. Viruses have various forms that attack plants. Some viruses are round like a ball or isometric, some viruses are rod-shaped, viruses are flexible rod-shaped, some are thread-shaped, some are bullet-shaped and some are gemini or flower shaped. Symptoms of virus attack on plants can be differ depending on the insect vector that attacks the plant (Sari, et al. [6]). Insects can play a role in transmitting viruses to plants and insects which transmit viruses to plants such as aphids (Aphid gosypii), whitefly (Bemiisia tabacai), aphids (Myzus persicei), banana fleas (Pentaloni nigronervosa), Fleas that attack the leaves of citrus plants and rose leaves (Aleurocanthus spiniferus), lice that attack the coconut leaves (Aleurodicus destructor Mask), Aphids on sugar cane plants (Oregma lanigera Zehntn), scale lice that attack coffee plants and crops cloves (Coccus viridis) and dompolan lice (Pseudococcus citri Risso), Brown planthopper (Nilavarpata lugens), Green planthopper (Nephotettix virescens) which causes tungro disease in rice plants (Anggraini, et al. [2,3,6]). (Rahayu, et al. [7-11]).
Objective Evaluation of the Effects of Human-Robot Communication Using Gaze and Cerebral Blood Flow
Introduction
The birth rate is rapidly declining while the population is aging in Japan. The aging rate, which is the percentage of the total population older than 65 years, was 28.4% in 2019 and is expected to rise to 38.4% by 2065 (Cabinet Office and Government of Japan, [1]). This rapid population aging has caused a shortage of human resources and economic funding to provide adequate care for older adults (Muramatsu & Akiyama, [2]). To address this issue, the Ministry of Health, Labor, and Welfare in Japan has launched the “Promotion of the Development and Dissemination of Nursing Care Robots” project to support the introduction of robots and their practical use (Ministry of Health, Labor, and Welfare, [3]). The Ministry of Economy, Trade, and Industry (METI) and the Japan Agency for Medical Research and Development (AMED) launched the Robot Care Equipment Development Project to support the development of robots to solve problems in nursing care facilities (Ministry of Economy, Trade, and Industry & Japan Agency for Medical Research and Development Announcements, [4]). In medical and welfare fields, advanced technology is required to detect information, make judgments, and take action.
A robot that assists in nursing care is defined as an intelligent mechanical system that possesses the three elemental technologies of “sensing” (sensor system), “judging” (intelligence/control system), and “operating” (drive system). Consequently, the introduction of robots in the medical and nursing care fields is expected to be an effective countermeasure in an aging society with a declining birth rate. Humans engage in extremely complex communication, and a combination of verbal and nonverbal cues are used to effectively send and receive information to others. According to Birdwhistell [5], only 30–35% of meaning is conveyed through words, and the rest is conveyed through nonverbal communication. Mehrabian [6] also stated that only 7% of the attitude and personality tendencies of individuals are estimated using words; 38% and 55% are based on peripheral language and facial expressions, respectively. In addition, communication in medical care settings requires more advanced techniques than ordinary communication due to a combination of factors, such as the illness and age of a person.
If robots in medical and nursing care settings cannot make appropriate nonverbal expressions or understand the nonverbal responses of users, their conversations will feel unnatural, and good relationships will not be established with them (Mathur & Reichling, [7]). However, only a few studies have objectively evaluated the reactions of participants to using communication robots. An appropriate evaluation of eye gaze and nonverbal responses of humans will enable robots to be equipped with functions to compensate for unnaturalness in the future. Therefore, this study aimed to analyze the changes in gaze and cerebral blood flow during conversations between humans and robots to objectively evaluate their impact on humans.
Methods
Study Design
The study was conducted from April to December 2022, and the participants were three nursing students in their 20s and 40s at a university. PALRO (Fujisoft) was used as the communication robot (CR). Before the experiment, adjustments were made to set the eye level to that of the CR, and the distance between the CR and the participants was 50 cm. The participants wore a HOT-2000 (NeU Corporation) brain activity meter and had a 15-minute conversation with the CR after resting for 15 min in a room separate from the experimental room. The participants were asked to speak naturally using the same dialogue program built into the robot when it was sold. During the conversation, gaze information was collected using a gaze analyzer (Tobii Technologies), and changes in heart rate (HR) and cerebral blood flow (HbT: total hemoglobin [Hb] change (left/right)) were measured using the HOT-2000. Video recordings were used for subjective evaluations (Figure 1).
Figure 1
Ethical Considerations
This study was conducted with the approval of the ethics committee of the institution of the researchers (No. 4221). No harm to the participants was observed. The participants were notified that their privacy would be protected.
Previous Studies on Eye Gaze
We used a Tobii Eye Tracker 4C from Tobii Technologies (150 eye movements/s) to perform gaze analysis. The Tobii Eye Tracker 4C is a noncontact device that detects the gaze of a person using infrared reflections, and is currently used in home video game consoles. It has been used in a nursing study that reported differences in how nurses gaze at electronic medical records (Takami, et al. [8]) and intravenous injections (Sugimoto, et al. [9]) depending on their clinical experience.
Previous Studies on Cerebral Blood Flow
The HOT-2000 meter was developed by the NeU Corporation. It is used to measure cerebral blood flow, and it uses light at a wavelength that is easily absorbed by hemoglobin (approximately 800 nm) based on the principle of optical topography. When brain regions on the light path are activated, blood flow and light absorption increase, allowing for the non-invasive evaluation of brain activity. The system can be used only by attaching a headset; therefore, data collection can be performed with minimal burden on the participant. It has been used in medical studies and has shown the relationships between brain oxygenation levels during sleep deprivation and poor cognitive performance of nurses during night shifts (Durán-Gómez, et al. [10]). It has also been used to study the effects of sex on brain activity during mental workload (Keshmiri, et al. [11]), limitations of near-infrared spectroscopy (NIRS) data analysis (Keshmiri, et al. [12]), and the relationship between cerebral blood flow and mental stress using NIRS (Komuro, et al. [13]). Figure 2 shows a conversation between a human and the CR.
Figure 2
Statistical Analysis
The gaze data were divided into six (2 × 3) locations, with (X, Y) = (0, 0) as the coordinates of the center of the participant’s gaze range (the central part of the CR face), and descriptive statistics (frequency and percentage) were obtained. Cerebral blood flow was divided into that before the conversation, during the conversation, and characteristic scenes (scenes in which participant smiles and laughter were extracted). Welch’s one-way ANOVA was analyzed, followed by the Games-Howell method for post-hoc testing. Statistical significance was set at p-values of <0.05. All statistical analyses were performed using SPSS for Windows (version 20.0; SPSS Inc., Chicago, IL, USA).
Results
The results of the gaze analysis showed that Area 2 (n=2016, 44.15%) was the most frequent for Participant 1, followed by Area 3 (n=1708, 37.41%). For Participant 2, Area 2 (n=14606, 96.44%) was the most frequent, followed by Area 3 (n=362, 2.39%). For Participant 3, Area 2 (n=21750, 94.07%) was the most frequent, followed by Area 1 (n=680, 2.94%) (Figure 3). The changes in cerebral blood flow and heart rate are shown below. The changes in the right cerebral blood flow in Participant 1 were highest for the characterized scenes (0.91±0.14), followed by during (0.87±0.18) and before (0.50±0.15) conversation, with significant differences for all scenes (p<0.05). The changes in the left cerebral blood flow were significantly different (p<0.05). The highest value was obtained before the conversation (-1.47±0.21), followed by that for the characteristic scene (-1.57±0.12), and that observed during the conversation (-1.58±0.13). The heart rate changes during conversation (83.18±5.67) were significantly higher than before conversation (80.47±14.88) (p<0.05), and those occurring during the characteristic scenes (83.11±5.98) were significantly higher than those before the conversation (80.47±14.88) (p<0.05).
Figure 3
The changes in the right cerebral blood flow in Participant 2 significantly differed (p<0.05) for the characterized scene (0.62±0.07), during the conversation (0.23±2.37), and before the conversation (0.15±0.14). The changes in the left cerebral blood flow were also significantly different (p<0.05) for the characteristic scenes (0.53±0.24) and during (0.10±2.21) and before (0.01±0.10) the conversation. The heart rate changes were higher during the conversation (81.47±5.76) than for the characteristic scenes (78.41±2.78) and before the conversation (73.91±3.37) (p<0.05). The changes in the right cerebral blood flow in Participant 3 differed significantly (p<0.05) before (-0.33±0.09) and during (-0.46±0.12) the conversation and for the characterized scenes (-0.52±0.13). The changes in the left cerebral blood flow were higher during the conversation (0.09±0.10) than for the characteristic scenes (0.07±0.10) and before the conversation (-0.38±0.18) (p<0.05). The changes in the heart rate were higher for the characteristic scenes (74.97±3.24) and before (73.66±2.60) and during (73.09±3.48) the conversation (p<0.05) (Table 1).
Table 1: Welch’s Analysis of Variance Results for robot and human conversation.
Note: We1ch’s Analysis of variance, abbreviations: SD, standard deviation, RtHb: Right to Homogrobin, HR: Heart Rate, *p<0.05, **p<0.01, ***p<0.001. Post hoc test Games-Howe.
Discussion
The gaze analysis showed that the participants most frequently gazed at the periphery of the face of the robot. This means that basic communication skills, such as making eye contact with another human (or patient) and gazing around the face (University of Minnesota, [14]), were used during conversation with a robot. However, PALRO, a CR, is designed to show various responses with 60 LED lights placed around its face, instead of nonverbally demonstrating pleasure, anger, sadness, or pleasure like a human face. Therefore, it is possible that the participant was trying to obtain response information by gazing at the area around the face of the PALRO (FUJISOFT website, [15]). The cerebral blood flow values were the highest for participants 1 and 2 during the characterized scenes. Laughing during conversation is considered an emotional change and may affect cerebral blood flow. Sugawara et al. [16] stated that laughing causes changes in blood pressure and heart rate, resulting in temporary but beneficial changes in the vascular system. The ability of a robot to stimulate emotional changes in humans is considered effective in maintaining cognitive function, and robot use in facilities for older adults is expected to have benefits.
Studies have shown that robotic interventions have improved the state of asthenia in patients with dementia (Valentí Soler, et al. [17]). However, the left cerebral blood flow changes varied depending on the participant. The involvement of the right brain has also been studied. Brain activity changes varied when the participant was engaged in conversation, as the left brain generally concerns language functions during conversation (Taylor & Regard, [18]). It is possible that the value changed significantly for the scenes during which the participant actively spoke to the robot, and it is necessary to scrutinize the content of the conversation and the number of statements made. Heart rate is known to fluctuate with psychological factors, and transient changes occur with excitement and pain, among others (Levy, et al. [19]). The heart rates of participants 1 and 2 were significantly higher during conversations. Communication with a robot is considered to affect human autonomic nervous system activity, leading to changes in heart rate. Based on the above, conversations with robots can be objectively evaluated for their effect on human cerebral blood flow, heart rate, and the autonomic nervous system activity that controls them. Starting a conversation with a robot may effectively prevent lonely older adults from decreasing their activities and the decline of ADLs and function. We will continue to conduct experiments under the same conditions on the older adults and evaluate the results.
Limitations
While changes in eye-gaze frequency and cerebral blood flow were measured, it is difficult to directly compare a robot and a human in an experiment. It is also difficult to maintain similar conversational content across all participants.
Conclusion
In this study, we focused on human-robot conversational situations and objectively evaluated the impact of communication with robots on humans. The results showed that even if the other party was a robot, humans frequently gaze at the area around the face. The changes in cerebral blood flow showed differences; blood flow increased significantly for scenes involving laughter relative to the others. While addressing problems such as human resource shortages, robots may also have a positive effect on humans by stimulating conversations and increasing human activity such as recreation, conversation, physical exercise etc..
Acknowledgments
We would like to express our sincere gratitude to the participants of this study. This study was supported by JSPS KAKENHI (grant number: 19K1973500).
Insect-Based Diet, A Promising Protein Source for Monogastric, Affects Human Health and Poultry Performance: A Case Study on Black Soldier Fly Lavae
Introduction
As a means to increase productivity through improvements in health, feeding and daily management poultry should explore alternative nutrient resources especially for economically challenging nutrients like proteins and energy [1]. However, for most of the crop waste and industrial residues have the key nutrients complexed with inert compounds like lignin making the unavailable to non-ruminant livestock and poultry [1]. On the other hand, nature has provided readily available resources inform of insect based ingredients particularly black soldier fly larvae have been approved to be used as feed in monogastric livestock especially poultry piggery and fish [2]. Defined as a protein rich larvae meal is a protein-rich feed ingredient Black soldier fly lavae meal can replace the fish meal in both monogastric livestock and aqua feeds as it competes favorably with plant based sources like lablab [3]. This is attributed to the fact that the demand and price for the protein feed ingredients and fish meal increase in the market, necessitates the need for alternative protein feedstuff used as a substitute for the fish meal [3]. From the various studies conducted, it can be concluded that the black soldier fly larvae meal can effectively replace fish meal up to 50% in the fish feed without having any adverse effect on the fish body [4]. Thus, the use of black soldier fly larvae meal as an alternative source of protein can reduce the production cost in the aquaculture industry without reducing fish’s quality. Besides being a promising fish meal substitute monogastric livestock species on the other hand reproduced appealing carcass composition, antioxidant enzyme activities, as well as digestive enzyme activities [5].
Whereas 50% replacement of fish meal with black soldier fly larvae meal in the diet showed positive effects without causing adverse effects on growth and feed utilization parameters several authors reported negative effects beyond 75% replacement [1,2]. Insects have been found to improve animal health. The chitin, antimicrobial peptides and fatty acids that are contained in insect meal have been reported to boost immune response in livestock [3]. (Xiao, et al. [4]) reported an increase in immune response of fish fed diets in which FMP was substituted with BSLMP through increased serum lysozyme activity. (Park, et al. [5]) reported a high antibacterial activity of black soldier larvae extracts in a study were the larval extract was tested on a broad spectrum of bacteria species. However, the dearth of information regarding effects of black soldier fly meal substitution of public health warrants further investigation.
Defined as fast growing avian species broiler as compared the egg type chickens and therefore correspondingly have higher protein requirements ranging between 18 and 23% on dry matter basis depending on age they offer the best case study on assessing the feeding values of protein substitutes in monogastrics. Since optimal growth is only attained when amino acids are provided in their adequate and right ratios [6] for example lysine and methionine should be maintained in a ratio of about 1:2.5 [7]. It implies that it is not just the protein content of the feed but the amino acid composition that is most crucial in monogastric nutrition [5]. Therefore, the quality of protein and hence its biological value is determined by the way essential amino acids are balanced. Specifically, as reported by several authors, increasing the ratio of sulphur amino acids to lysine from 50 to 77 increased weight gain in broiler chickens [8]. Such responses are attributable to the fact that amino acid profile of diet affects feed intake, feed utilization efficiency and body weight gain in monogastric livestock [9]. In addition, slight deficiencies in dietary methionine trigger detrimental reduction in feed intake and growth of birds [5,10] while optimizing sulphur amino acids improves growth and feed utilization [11]. Therefore, balancing monogastric diets does not only call for providing adequate protein content but also the amino acids and their ratios.
Ingredients used as protein sources in poultry diets differ in their amino acid profiles (composition) and thus their quality. For example sulfur amino acids and lysine are the most limiting in plant protein sources but abundant in animal protein sources [12]. Reduced growth performance and feed efficiency has been reported in studies where chickens were fed on diets without an animal protein source [13]. Therefore, animal protein sources should be considered while formulating poultry diets so as to balance the amino acid profile and thus improve growth performance and feed efficiency of birds.
Alternative Protein Sources to FM in Broiler Chicken Diets
Protein as a nutrient required by broiler chickens is supplied by various ingredients majorly cotton seed cake, soybean meal and FM which have an impact on the quality of protein in the diet and thus the performance of birds. Fish is and has always been the major animal protein source in broiler diets [14]. Fish has a high crude protein content and well balanced amino acid profile [14] and is a good source of phosphorus and calcium [6]. However, its availability for inclusion in poultry diet is limited by the several users of the product, thus a need to explore other animal protein sources that can be used as alternatives. Other ingredients used to substitute fish include both plant protein sources and animal protein sources [7]. In addition to the fact that a few plant protein sources have been explored as substitutes for FM, reduced performance has been reported when plant protein sources were used to substitute FM due to imbalances in the amino acid profile and high fiber content [8]. Among the animal protein sources that have been used to substitute fish include earth worm and insects from several orders [14,9]. However, studies have not fully exploited the use of these nonconventional protein sources either as sole protein sources or as substitutes for FM. Therefore, there is need for further research on use of insects as substitutes for FM.
Insects have been identified as a potential source of protein in poultry rations many years ago [10] and their use is still investigated up to date [11]. Several insect species under a number of orders have been studied for their potential to feed poultry [11] with order Diptera being the most widely studied. Insect species studied and so far used as protein sources under the order Diptera include house fly (Musca domestica) [10,11], blow fly (Chrysomya megacephala) 13 blue-bottle fly (Protophormia terraenovae) among others. Despite the potential of BSLM reported in previous studies questions still arise on the quantity that can substitute FM, effect on growth, feed utilization, feed cost, carcass quality and poultry physiology.
Nutritional Composition of Insect Meals Used for Animal Feed
Protein is one of the key and most expensive feed nutrient in non-ruminant livestock nutrition. With numerous insect species having an equivalent protein content as fish [12], insect based feed ingredients are a good source of proteins. Interestingly, other insect based proteins have even more protein content than fish [6,15,16]. Specifically, several authors have reported house cricket meals, field cricket, house fly maggots, grass hopper and black soldier flies meals to contain 60.4%, 58.3%, 60.4%, and 60% crude proteins, respectively [17-20]. Therefore, since some insects like silk worm larvae have substituted fish without adverse effects on nutrition related performance of the monogastrics especially poultry and piggery [21], the high crude protein content makes many insects a potential source of protein in poultry diets. However, with no amino acid synthesis mechanisms, monogastric livestock, poultry and fish utilize the primary building blocks of proteins. This implies that the quality of proteins in monogastric and poultry feeding perspective primarily depend on the sequence of the amino acids as well as their bioavailability [5]. Interestingly, basing on the protein biological value, insect larvae especially from order diptera have high quality protein that compares well with that of fish and soybean meal [7,12]. (Wang, et al. [22]) noted a higher amino acid profile of field cricket than that of fish with an exception of histidine, whereas [21] also found silkworm to be superior in most essential amino acids than fish meal. In comparison to fish meal, insect meals are deficient in histidine, lysine and threonine amino acids [7] but seem to have a higher content of tyrosine and valine [23].
Razak et al also found the house cricket to have more tryptophan, tyrosine and valine amino acids than soybean and fish [24]. The most limiting essential amino acids, lysine and methionine were found to be higher in the maggot meal (6.04% and 2.28% respectively) when compared with those of other conventional protein sources including fish meal [25] (Aniebo, et al. 2008. When compared with fish meal, black soldier fly lavae meal contains higher quantities of histidine and valine but lower levels of methionine, threonine and cysteine [19,26,27]. Despite the lower levels of some amino acids, previous researchers have reported black soldier fly lavae meal to have sufficient crude protein and amino acid for broiler chickens [7,28]. Therefore, regardless of the quality of protein portrayed by insect meals the need for supplementation with other protein sources or with synthetic amino acids that could be deficient in insect meals in case insect meals are to be used as feed or feed ingredients is indispensable. Besides proteins, metabolizable energy plays a complimentary role in protein utilization especially during protein synthesis. For the case of insect meals, the energy component is largely contributed to by the crude fat fraction of the ingredient which varies with insect species [7]. Generally, insects have a high fat content of over 70% more than that of fish meal [9,11].
The high crude fat of larval insect meals is as a result of the extra deposition of fat during the larval stage of some insect species for use by the adults that do not feed due to absence of mouth parts. Specifically, black soldier fly lavae meal contains a crude fat content that ranges between 14% [29] and 39% [23], mealworm over 28% [26], housefly maggot meal 24% [30] among others. Furthermore, the fatty acid profile of insect larvae differs from that of fish meal [31]. Insects, black soldier fly lavae meal inclusive have higher levels Saturated Fatty Acids (SFA), high omega 6 and over three times lower omega 3 fatty acids as compared to FM [32,33]. Lauric acid is the most abundant SFA in black soldier fly lavae meal [31]. However, the high levels of Crude fat accounts for the reduced feed intake in insect meal based diets which leads to inadequate supply proteins and other nutrients. This implies that, the difference in the lipid profile of insect meal from that of fish meal can alter the dietary lipid profiles when used to substitute fish meal not only affects livestock and fish but also effects human nutrition health.
Factors that Affect Nutritional Composition of Insect Meals
Insect meals differ in their nutritional and mineral content and such variations are attributed to species differences, substrate or diet of the insect, age at harvest and method of drying and processing [34,35]. The growing substrate has a major influence on the nutrient composition of the resultant insect meal. The nutritional content of the substrate especially the crude protein influences the crude protein content of the insect larvae [23]. (Spranghers, et al. [36]) reported a 5% rise in crude protein and a 77% increase in crude fat of black soldier pre-pupae fed on substrates with correspondingly higher crude protein and crude fat respectively. Ramos-elorduy [9] also reported a difference in crude protein content of mealworm larvae with diet. On the other hand raising housefly maggots on different substrates each containing a proportion of poultry droppings did not change the crude nutritional composition of the resultant maggots [37]. The observed results could have been a result of the small variation in substrate composition. Therefore, the material selected for production of insect meals is very important as far as the nutritional composition of the resultant larvae is concerned. Nutritional composition of insects also differs with age at which they are harvested and the method used to process the insects or insect larvae [34].
Generally, the crude protein content of insect larvae reduces as they grow while the crude fat increases [7]. As the larva develops towards the pupa stage it stores more energy in form of fat which it utilizes in the later adult stage. Some adult insects for example BSF do not have mouth parts and thus do not feed but survive on the fat stores [38]. Aniebo and Owen [39] reported an increase in fat content and decrease in crude protein content of housefly larvae (Musca domestica Linnaeus) with age. (Singh, et al. [34]) reported higher concentrations of amino acids in the meal of blowfly maggots harvested one day after hatching than those harvested later. On the other hand, earlier studies reported a change in the composition of insect meals exposed to different processing procedures. Higher protein content (50.9%) and less fat (22.8%) was reported when maggots were oven dried as compared to sun dried maggots (47% CP and 26.4% CF) [7]. Therefore, variations in the nutritional content of insect larvae due to age and processing methods should be considered while choosing to incorporate insects in poultry diets.
Health Associated Benefits and Risks in Humans
Meat is the major source of fat especially SFA in human diet. Saturated Fatty Acids have been associated with some diseases like cardiovascular diseases and some types of cancers [40]. The incidence of such diseases is associated with both the amount and type of fat consumed. Therefore, there is need to assess the quality of the meat for human consumption in order to address the latest health concerns. Despite the number of studies conducted to assess the effect of inclusion of insect meals in diet of broiler chickens, results stop at describing effect on growth performance. Health benefits derived from meat are majorly dependent of the meat fat and fatty acid composition. Cardiovascular diseases are correlated to the level of Low Density Lipoprotein (LDL) cholesterol. Diets high in SFA for example lauric, myristic and palmitic acids contribute to the increase in both LDL and HDL cholesterol level while oleic and linoleic acids are said to increase HDL and lower LDL [41,42]. On the other hand Monounsaturated Fatty Acid (MUFA) and Poly Unsaturated Fatty Acids (PUFA) in particular long chain n-3 PUFA reduce the risk of coronary heart disease in humans through their effect on the level of LDL [43]. Further, lack of n-3 PUFA or an imbalance of n-6 and n-3 fatty acids in the diet can result into increased inflammatory and immune disorders [43,44].
Some fatty acids like linoleic, linolenic and arachidonic are essential fatty acids that should be supplied in the diet since the body cannot produce them. Strategies to modify the quality of meat through dietary modifications have been adopted of recent. In such strategies ingredients that are considered beneficial to human health have been added to diets of different animal species [45], after different studies have proved that the composition of the diet affects the resultant meat composition [46] Un like ruminants, in poultry dietary fatty acids are absorbed unchanged before incorporation in tissues and thus the content of the different fatty acids of muscles increase through increasing their content in the diet [47]. Plasma triglycerides and total cholesterol can be used in prediction the amount of carcass fat. Apart from the age of birds dietary composition also affects the plasma lipids [48,49]. For example including sugar beet pulp in diets of broiler chickens reduced the total serum cholesterol concentrations of the birds [48]. Inclusion of garlic in diet reduced the serum levels of cholesterol, low density lipoprotein and triglycerides but increased high density lipoprotein levels [46]. Insect meal, BSLM in particular has been reported to reduce plasma triglycerides and total cholesterol in chickens [4,50]. Dietary fat sources high in SFA increase plasma triglycerides and total cholesterol in poultry. Substituting FM with grass hopper meal increased total cholesterol in rabbits [18].
Utilization of BSLM in Livestock Feed and Impact on Growth, Feed Intake and Utilization
The BSLM is a potential protein source in fish [28,33], pig [36] and poultry rations [51]. Studies have assessed its potential either as a substitute for FM [51], soybean meal [52] or as a sole protein source [4,39,53]. Additionally BSLM has also been assessed as a fat source in broiler diets [39]. Despite the potential of BSLM as a protein source in poultry diets, effects of its inclusion at higher levels in broiler chicken diets are not known.
Different inclusion levels and forms of BSLM have been studied as feed or feed ingredient for livestock. BSLM has been added at lower levels in broiler chicken diets, the highest level being 50% in starter diets [54] and 55.5% in finisher diets [54]. However, with other poultry species up to 100% inclusion has been studied [54]. Some studies have assessed levels up to 15% only in broiler chickens and quails [39,55] in pigs levels of up to 8% inclusion have been assessed [36]. While in fish diets levels more than 50% have been studied [38]. In many of the studies the larval stage of BSF has been studied and in the processed form as a meal [56]. In some studies further processing of BSLM was done by either partially or fully defatting the meal [18]. Performance of livestock fed on diets containing BSLM varies with inclusion level, method of processing and stage of growth. Inclusion of up to 50% BSLM in fish diets did not affect feed intake and the feed conversion Ratio (FCR) but reduced weight gain in rainbow trout [57].
On the other hand, increasing the level of BSLM beyond 50% reduced feed intake, FCR and weigh gain of different species of fish [28,58]. In pigs, a higher feed intake was observed for a diet that contained BSLM compared to a soybean based diet [59], but lower levels up to 8% did not affect growth in weaned piglets [36]. Incorporating up to 50% BSLM in diets of quails increased feed intake by 7% and egg weight by 9% [60]. While complete substitution of soybean in diets of laying hens reduced feed intake, percentage lay, egg weight and egg mass [4]. Previous findings report similar or an increase in feed intake, FCR, growth performance and feed utilization of diets containing less than 55% BSLM [51,54,55,61]. On the other hand substituting soybean up to 50% with BSLM in broiler chicken diet reduced feed intake and weight gain when the amino acid profile of the diet was not balanced, however, balancing the amino acid profile increased feed intake and weight gain [29]. Partial defatting of BSLM in addition to increasing the CP content and digestibility of the meal, also improves performance among broiler chickens [18].
In conclusion, lower levels of BSLM have proved to be a potential protein source specifically in broiler chicken diets through improving performance. Several studies have reported a high digestibility of BSLM which increases the potential for utilizing the larvae meal in poultry diets. Apart from quails where a low (<50%) CP digestibility of a diet containing BSLM was reported [29] studies with fish [28], pigs [36] and chickens [55] have reported higher (>70%) CP digestibility of diets containing BSLM. The digestibility of BSLM was higher than that of mealworm among broiler chickens [29]. In addition, digestibility of BSLM is affected by method of processing [53]. In the same study BSLM dried at 1000C and that which was defatted had higher digestibility coefficients than full fat BSLM dried at 650C. Weaned piglets digested defatted BSLM better than full fat BSLM [36]. In addition, presence of chitin in BSLM could reduce digestibility of diets in which it is included [48]. Despite the higher digestibility of BSLM that has been reported, factors like the high fat content of the larvae meal, presence of chitin and drying temperatures could affect digestibility of diets in which BSLM is included.
Effect of Diet on Immune Response of Livestock and Poultry
Immune response is the number one mechanism used by the body to fight against harmful organisms. The immune system can be categorized as humoral immunity which is mediated by the bursa dependent component by macromolecules such as antibodies or cell mediated immunity which involves the activation of phagocytes, leukocytes or White blood cells (WBC) majorly controlled by the thymus [62]. WBC are produced in the bone marrow, liver and lymphoid tissues which include the bursa, spleen and thymus [63]. Types of WBC include monocytes, lymphocytes, heterophils, eosinophils and basophils. Control of NCD and IBD in chickens starts right from proper health management of the parent stock. The parent stock at hatcheries is vaccinated against all diseases and thus chicks possess high levels of antibody at the time of hatching which they obtain antibodies from their parents. The maternal antibody level reduces thereafter with age and disappears by day 21 [34]. Immune response increases with age and reduction in maternal antibody in chicks. The reduction in antibody titers against inactivated NCD virus during the first week of age was attributed to the interference by maternal antibodies [64]. (Grozdanić, et al. [64]) reported the highest immune response on day 21 when maternal antibody had disappeared and lowest on day one when maternal antibody was highest. Dietary composition especially medicinal ingredients affect immunity of birds.
(Rahimi, et al. [65]) reported an increase in antibody titers in response to sheep red blood cells but not to new castle disease vaccine when chickens were fed on coneflower extract. In addition, supplementing the diets of broiler chickens with chromium methionine increased antibody titers against both NCD and Infectious Bronchitis Virus [66]. Including the sun mushroom in the diet of broiler chickens increased the percentage of eosinophils [67]. Heterophils are said to range from 30 -75%, lymphocytes 20 – 65%, monocytes 0-5%, basophils 0-5% and eosinophils 0-4% [67]. It has been demonstrated that the numbers of leukocytes in avian species change with physiological changes, disease and stress [68]. Dietary composition especially the fatty acid composition has an influence on the immune system of birds [69]. Fatty acids and their derivatives are known antimicrobial, antifungal and antiviral agents, promote development of lymphoid tissues and increase lymphocyte proliferation [70,71]. Researchers have studied the effect of fatty acids on the immune system and findings confirm the antifungal, antiviral and antimicrobial effect of different fatty acids [72]. Antiviral fatty acids at low concentrations act by affecting the cell envelope causing leakage while at higher concentrations they completely disintegrate the envelope and cell particles or even the cell membrane causing death [69]. Among all fatty acids lauric acid has been the most widely studied and reported for antibacterial, antifungal and antiviral activity [73-75].
In addition feeding chickens on diets containing lower omega 3 fatty acids increase lymphocyte proliferation [72]. Insects have been found to improve animal health. The chitin, antimicrobial peptides and fatty acids that are contained in insect meal have been reported to boost immune response in livestock [51]. Xio, et al, [76] reported an increase in immune response of fish fed diets in which FMP was substituted with BSLMP through increased serum lysozyme activity. Park et al reported a high antibacterial activity of black soldier larvae extracts in a study were the larval extract was tested on a broad spectrum of bacteria species [77].
Effect of Diet on Gut Colonization, Structural Development and their Impact on Nutrient Digestibility and Health
At the time of hatching, chicks are exposed to microbes that exist at the surface of the egg shell. This originates both from the mother chicken gut and the environment. The microbial population continues to change and its composition and numbers is influenced by diet of birds [78,79] among other factors. Dietary factors that influence gut micro flora include the ingredient composition of feed, digestibility of feed, protein content and amino acid content of the diet and protein source [78,79] fatty acid profile [35]. The populations of Clostridium perfringens in the ileum and caecum increased with the level of protein content when birds were fed FM, meat meal, feather meal or potato protein concentrate as a sole protein sources but not with soybean meal, corn gluten meal or pea protein concentrate [78]. Gut microbial population changes with age and section of the GIT [80,81]. In addition the source of crude protein in the diet affects microbial population [78]. In the study, the population of Clostridiun perfringens in hind gut significantly increased when birds were fed FM instead of soybean meal. (Spranghers, et al. [35]) reported a reduction in streptococci and lactobacilli when weaned piglets were fed a diet containing 8% BSLM substituting soybean meal. Feeding laying chickens on a diet with BSLM as a protein source increased both the microbial diversity and abundance in the caeca compared to a soy bean meal based diet [50]. The caecum contains more bacterial population in terms of number than the ileum [82]. An adult chicken GIT is inhabited by up to 1013 bacteria [83].
The population grows rapidly within the first week post hatching. Previous studies reports the population to plateau within the first four days after hatching, however some changes occur throughout the entire life of broiler chickens [83]. One day post hatching, the bacterial numbers in the proximal and distal parts of the ileum is expected to be 108 and 1010 cells/g of digesta respectively, the maximum population of more than 109 in the ileum and 1011 in the caecum is obtained in less than seven days and there after the population of bacteria remains stable up to 30 days [83]. Bacteria in the gut of chickens have a positive effect on gut morphology and nutrition. Gut micro-biota help in digestion and synthesis of dietary compounds, are involved in gastrointestinal development, regulate intestinal epithelial proliferation, host energy metabolism and synthesizes vitamin K and most water soluble vitamins like biotin, cobalamin, folate nicotic acid pantothenic acid, riboflavin and thiamine [83]. Gut miro-biota of chickens also impact the immune system and the health of the chickens in general. Bacterial species in the gut keeps changing in composition and number until a stable ecosystem is obtained. Once a stable ecosystem is obtained bacterial subsequent growth occurs in synchrony with digesta passage rate keeping the bacteria density in each part of the GIT constant. The more stable the gut micro biota the more the bird can resist infections especially in the gut [50]. Stability of the gut bacterial population prevents colonization by pathogenic organisms. Micro-biota has an influence on the immune system through its influence on the intestinal wall, regulates intestinal epithelial proliferation, is involved in inflammatory immune responses and fills the microbiological niche that would in their absence be occupied by harmful enteric micro-organisms [83]. Bacteria in the gut provide a physical barrier to the gut epithelia from harmful invading micro-organisms. Gut microbes also produce short chain fatty acids which are bacteriostatic and some generate bacteriacins. Adding insects in diets of insects has proved to modulate gut microbes in chickens and improving immunity [84]. Dietary composition influences structural development of gut. An increase in jejunal villi, crypt depth and a reduction in goblet cell number was observed when broiler chickens were fed diets with varying level of methionine [85]. An increase in the villi height was observed in rats fed monounsaturated and polyunsaturated fatty acids compared to those fed saturated fatty acids. Feeding broiler chickens on a diet containing BSLM did not affect the structure of the duodenum and caecum with respect to villi length and crypt depth but increased the jejunum villi length by 17%. Therefore, the amount and composition of dietary fat affects the structure poultry gut, of which the composition of crude fat is influenced by the ingredients supplying the fat.
Conclusion
The high crude protein content of insect meal based diets justifies utilization of the larvae meal as a substitute for fish meal in poultry diets. However, the differences in amino acid profile from that of soybean and fish meal, presence of chitin, high crude fat content associated anti- nutrients and mycotoxins [5,86] could have an impact on feed intake and utilization hence the general performance of livestock and poultry and hence meat quality. In addition to affecting performance, attributes of insect based meals especially black soldier fly meals for example antimicrobial peptides [87] and the fatty acid profile gives BSLM the anti-bacterial, anti-viral and anti-fungal [77] activities could improve the immune response of birds. In general effects of BSLM on performance, meat quality, gut integrity and microbiology and immune response of birds is not fully studied especially at higher inclusion levels.
Data and Information Sources
The information utilized in this review came from manuscripts that were just published in a variety of journals. Electronic data sources like the Directory of Open Access Journals (DOAJ), Research Gate, Web of Science, Science Direct, Google Scholar, and PubMed were used to access databases. Furthermore, other pertinent articles were found by searching through the citations included in the articles from the databases. The search engines were used to find “poultry,” “edible insects,” and “insect meal.”
Impulse Oscillometry and Exercise Bronchoconstriction in Children and Adolescents
Introduction
Exercise-induced bronchoconstriction (EIB) is defined as the transient and reversible narrowing of the lower airways that appears with physical exercise, which can occur in the presence or absence of bronchial asthma [1]. In children over 6 years of age, EIB is confirmed with the exercise challenge test (ECT) that measures the values of forced expiratory flow in the first second (FEV1) at 3, 5, 10, 15, and 30 minutes after at least 6 minutes vigorous exercise on a treadmill or cycle ergometer. A decrease equal to or greater than 10% in FEV1 compared to the baseline value before exercise confirms EIB [2]. Figure 1 shows an example of a traditional exercise challenge test with FEV1. Children under 6 years of age have shorter expiratory times and less ability to perform conventional ECT. In this group of children, it has been recommended to perform ECT after a free run and use the forced expiratory volume at 0.5 seconds (FEV 0.5). A 13% drop in FEV 0.5 ha has been proposed to consider BIE in preschoolers aged 3 to 6 years who perform spirometry. The spirometric maneuver is effort-dependent and requires important cooperation on the part of the patient, who must inhale at maximum capacity and exhale with the greatest force and duration possible. On the contrary, Impulse oscillometry (IOS) only requires minimal effort to perform inspirations and expirations, since they are performed at tidal volume. It has been shown that only 80% of children can perform the forced spirometric maneuver, a percentage that decreases at a younger age, unlike 100% of children who are able to perform the IOS even from 2 to 3 years old [3]. The purpose of this mini-review is to review available evidence for the use of IOS in the diagnosis of EIB.
Figure 1
Methods
A review of the evidence was carried out scientific study on the use of IOS in the diagnosis of exercise-induced bronchoconstriction. The search for articles was carried out between January and February 2024 in the databases Medline (PubMed), Web of Science (WOS), EBSCO Host, Science Direct, and SCOPUS. MeSH terms and free terms in their English version were used. The terms were grouped into two dimensions:
i) Impulse oscillometry
ii) Exercise-induced bronchial obstruction. The boolean operator was used “and ‘’ to integrate the three dimensions. The articles found were grouped into two categories by age range:
1) Preschoolers
2) Children and adolescents.
Preschoolers
A study carried out with children from 3 to 7 years old who underwent a free run of 6-8 minutes followed by measurement of IOS parameters at 2, 5, and 10 minutes after exercise, demonstrated that those who had a history of wheezing in the last 12 months showed changes in resistance at 5 Hertz ( R5), in the reactance at 5 Herz (X5) and in the Resonant Frequency (Fres) in comparison with non-atopic healthy controls. In this study it was confirmed that the most important changes occurred 2 minutes after finishing the exercise and that an increase in R5 over 35% can be considered an abnormal response to exercise, suggesting EIB [4]. Another study conducted in preschoolers with EIB measured by IOS and defined as an increase in R5 over 40%, demonstrated that the severity of EIB is correlated with higher levels of exhaled nitric oxide (FENO) in atopic than non-atopic children. Furthermore, FENO had a higher predictive value for EIB in IOS in atopics than in non-atopic wheezers, suggesting a different interaction between bronchial response and airway inflammation in non-atopic wheezers [5]. On the other hand, it has been shown that ECT with the free running of 6-8 minutes measured with IOS (R5 ≥ 40%) in children from 3 to 8 years old is capable of distinguishing children with probable asthma well. symptomatic of those with other conditions such as early recurrent wheezing, bronchopulmonary dysplasia, and healthy children [6]. A 6-month follow-up study carried out in preschoolers with asthma demonstrated that treatment with inhaled corticosteroids significantly reduces the percentage of BIE measured by IOS and defined as an increase in R5 ≥ 40% after exercise, so this parameter can also be used to monitor the effectiveness of the treatment [7]. Figure 2 shows an example of an exercise challenge test with IOS measuring R5.
Figure 2
Children and Adolescents
In a cross‐sectional study conducted on 74 children and adolescents with a previous diagnosis of asthma, aged between 7 and 17 years old, it was shown that AX, R5, and Fres had a moderate negative inverse correlation with FEV1 at 5 minutes after the end of the exercise (r = −0.69, r = −0.64 and r = −0.53, respectively, all with p < 0.05 ). In this study, cut-off points are also proposed in AX, R5, and Fres to discriminate between patients with and without EIB (FEV1 drop ≥ 10%,) however, the positive likelihood ratios were low and of little clinical relevance (LR+ 1.67, 1.75 and 2.08, respectively) [8]. Another study carried out in asthmatic children and adolescents aged 6 to 15 years showed that the parameter that best discriminates between patients with or without EIB confirmed by a 10% drop in FEV1 is R5 in IOS. In this study, the best cut-off point was found with increases in R5 at 30 minutes ≥ 14.1% of baseline (AUC = 0.74) with a low positive predictive value (PPV = 33.9%) and a high negative predictive value (NPV=97.5%), so the greatest use in this cut-off point is to rule out EIB if the R5 value is less than 14.1% [9]. A controlled study was recently published with 68 asthmatic or clinically suspected children and adolescents aged 6 to 16 years who reported symptoms with exercise. In this study, mean within breath differences in Resistance (Rrsexp- Rrsinsp) and Reactance (Xrsexp-Xrsinsp) were calculated, finding that the mean difference Xrsexp-Xrsinsp was higher in those with EIB than those without it, especially 18 minutes after exercise [10]. Figure 3 shows an example of a child who, after exercise, increases the reactance difference within the respiratory cycle [11].
Figure 3
Conclusion
Monitoring IOS parameters after exercise may be an alternative method to detect EIB when the patient cannot perform the conventional test with FEV1 measurement. In preschool IOS, increases in R5 above 35-40% have been shown to be effective for the diagnosis and monitoring of EIB. In children and adolescents, the cut-off point for R5 is lower and would be more useful in ruling out EIB than in confirming it. Differences in reactance within breathing are a promising alternative to diagnosing EIB in children and adolescents, which must be corroborated with more studies.
Conflict of Interest Statement
The author has no conflicts of interest to declare.
Funding Sources
The author has no relevant financial or non-financial interests to disclose.
Consideration of the Issue of Non-Invasive Diagnostics of Hormone Level in Athletes
Introduction
Saliva is one of the biological fluids produced by the human body [1-15]. A number of domestic and foreign studies have reliably established that the determination of markers of hormones and a number of other substances in saliva is an alternative method for determining the same substances in the blood serum of the subject [1,4,6-15,16,17]. A significant advantage of determining the level of hormones in the blood is its simplicity, accessibility, painlessness, and non-invasiveness. Also, the advantages of modern methods for determining the level of hormones in saliva is that, unlike blood sampling, saliva biomaterial can be taken at any time of the day, both during physical activity and during the athlete’s rest period, but with strict adherence to the conditions of the obtained biomaterial [1,4,12,16,17]. Naturally, an important point in the more active implementation of this method in practical clinical and sports medicine is the determination of standards for the content of a number of hormones (cortisol, cortisone, male and female steroid hormones, insulin, etc.) in saliva and the determination of correlation values for the level of these and other hormones in saliva, blood serum and urine. The technique of collecting material from the oral cavity does not require a private room, which is necessary when collecting urine and blood, i.e. this method is an alternative where collecting blood and urine is not possible [1,16,17,18].
According to research and practical experience cited by a number of authors, saliva samples are stable for 7 days at room temperature, 4 weeks at 2–8°C, and for a long period at temperatures below –20°C. Such high stability makes it possible to widely use saliva samples for analysis [1-3,6-9,17]. The problem that researchers continue to work on is the choice of reagents, preservatives, packaging material and its composition for collecting, storing and transporting saliva samples, as well as expanding the scope of application of this research method into practice, in particular in sports medicine, endocrinology and gynecology [1,4,7-20,19,20,21]. Domestic authors such as T.P. Vavilova, I.G. have been working quite long and fruitfully on the practical application of methods for determining hormones in saliva and all related aspects of this problem. Ostrovskaya, A.E. Medvedev, O.O. Yanushevich (2011, 2014) [2,3]. Under the leadership of T.P. Vavilova et al. A number of research papers have been published on the biochemistry of saliva and its research [2,3]. Of the domestic researchers involved in saliva diagnostics, I would also like to note the basic, classical works of such scientists as L.P. Gotovtseva, 2005; S.S. Mikhailov, E.V. Rosengart, 2008-2012; S.I. Piskov, 2008; S.A. Khaustova, 2010; L.V. Belskaya, O.A. Golovanova, V.G. Turmanidze, E.S. Shukaylo, 2011; V.A. Kurashvili, 2012; S.N. Didenko, G.D. Aleksayants, 2014; I.V. Evstigneev, 2014.
Among the foreign authors dealing with this problem, I would like to note the interesting studies of Vining R.F., McGinley R.A. (1986), reflected in the work “Hormones in saliva”, as well as in the works of Higashi T. (2012) “Salivary hormone measurement using LC/MS/MS: specific and patient-friendly tool for assessment of endocrine function” and Zolotukhin S. (2013) “Metabolic hormones in saliva: origins and functions” [15]. One of the latest works that deserves close attention, in our opinion, is the work of L.D. Hayes, N. Sculthorpe, B. Cunniffe, F. Grace “Salivary Testosterone and Cortisol Measurement in Sports Medicine: a Narrative Review and User’s Guide for Researchers and Practitioners” – “Salivary Testosterone and Cortisol Measurement in Sports Medicine: a Narrative Review and User’s Guide for researchers and practitioners” [15]. Classic works, especially regarding the introduction into practice of such an important index value as the “anabolism index” (AI), were introduced by S.K. Chang, H.F. Tseng, N.F. Tan, Y.D. Hsuuw, J. Lee-Hsieh, 2005 [12]. Also interesting are the works of such foreign authors as T.J. Cieslak, G. Frost, P. Klentrou, 2003 [12]; D.A. Edwards, K. Wetzel, D.R. Wyner, 2006 [14]; W.J. Kremer, A.D. Rogol, 2008 [19]. Here it is worth noting the earliest practical developments on this issue by S.K. Chang, H.F. Tseng, N.F. Tan, Y.D. Hsuuw, J. Lee-Hsieh, 2005, the authors of which laid the foundation for the use of saliva diagnostics in conducting research on testosterone, cortisol, their ratios, as well as describing the methodology of the saliva sampling process and conducting research procedures, which were taken into account and used in their works by other researchers this issue [12].
According to the authoritative opinion of such researchers as T.P. Vavilova et al. (2011, 2014), and V. Evstigneeva “In clinical and laboratory diagnostics, an important issue is the ratio of the concentration of steroid hormones in the blood and saliva” [2,3,17]. This is really of decisive importance, since thanks to the research of a number of domestic and foreign authors (S.S. Mikhailov, V. Rosengart, 2008, 2012; L.V. Belskaya, O.A. Golovanova, V.G. Turmanidze, E. S. Shukaylo, 2011), it was found that in saliva, unlike blood serum, a number of hormones, such as cortisol and testosterone, are in a free, unbound form, and their quantitative determination in the saliva of athletes is more reliable and informative [1,3,7,8,10-15,18]. This, taking into account the simplicity of collecting material and the ability to carry out express collection of material in any conditions and at any time of the day, makes saliva diagnostics an alternative and competitive one [1,3,6,10-12,14].
Results and Discussion
What hormones can be determined in a saliva sample? Today, many diagnostic laboratory centers determine the level of 17-OH-progesterone, androstenedione, DHEA, cortisol, progesterone, testosterone, free estriol, E2 estradiol [1,3,6-8,10-15,17,18]. From a physiological point of view, measurements of progesterone concentration in saliva can be used to monitor the menstrual cycle in women to determine the time of ovulation and to assess the function of the corpus luteum, which is very important in the early stages of pregnancy. Due to the constant fluctuations in progesterone levels, which also depend on the individual condition, it is very convenient to carry out several successive measurements of the hormone in saliva [4,9-21,16,18,19]. Control of estradiol and progesterone in the saliva of female athletes can not only help in controlling the menstrual cycle and its phases, both for building rational training and competitive micro, meso and macrocycles, but also conduct adequate express monitoring of the individual state of reproductive health of female athletes [1,6,7,12,15,16,17]. Human saliva and oral fluid contain the same hormones as blood serum (total and free thyroxine, total and free triiodothyronine, thyroid-stimulating hormone, cortisol, progesterone, prolactin, testosterone, follicle-stimulating and luteinizing hormones), but in significantly lower concentrations, in different at least for different hormones than in blood serum [1,3,6-8,10-15,18,17].
The concentration of progesterone and estrogen varies both in the blood and in saliva, depending on the phase of the menstrual cycle and the woman’s age [1,3,6-8,10-15,18,17]. Estradiol has a significantly higher concentration in oral fluid than in blood serum. There is a direct relationship between the concentration of hormones in blood serum and oral fluid or saliva [1,3,6,15-19,18,17]. Oral fluid hormones adequately reflect sex differences in serum hormones. In the blood serum and oral fluid, the concentration of estradiol, prolactin, FSH and LH in women is higher than in men, and in the luteal phase – progesterone [1,5,10,20]. In men, the concentration of testosterone in blood serum and oral fluid is many times higher than in women. In postmenopausal women, the concentration of estradiol and progesterone in the blood serum and oral fluid is reduced, and the latter was not detected in the oral fluid [3]. When determining dehydroepiandrosterone (DHEA) in the saliva of young female athletes (prepubertal, pubertal and adolescence), it allows us to determine such phenomena as delayed puberty or its premature manifestation [1,3].
Interest in determining the level of hormones at different age periods in athletes of both sexes, with their different levels of sports qualifications, as well as at different stages of physical activity, with different psychological components (in the pre-competitive, competitive and post-competitive periods), as well as during training and rest and rehabilitation – this is not a complete list of questions to which, in our opinion, coaches, sports doctors and psychologists, physiologists, and the athletes themselves would like to receive answers [1,16,17,20]. In this regard, L.V. Belskaya et al. (2011), rightly note that “The main reason for the negative impact of physical activity is the insufficient use of modern quantitative methods that allow for careful medical monitoring during the training process” [1]. It is important to monitor the dynamics of hormone formation during physical activity using strength, speed, complex coordination and other exercises. And here it is difficult to imagine an attempt by a researcher to collect blood and urine from athletes at all these stages. Taking a saliva sample appears to be a comprehensive alternative here [1,3,4,12,17].
Unfortunately, there is not yet a comprehensive practice of a widespread, fully algorithmized (standardized) procedure for this type of research, although there is a sufficient number of works on this issue [17]. When considering the methods used, the use of equipment and reagents, there is also no unity, since the authors carried out their studies using different methods, equipment and reagents [6-8,17]. What did the authors most often use in their research? So, for example, I.V. Evstigneev, when studying steroid hormones in saliva (testosterone, androstenedione, dehydroepiandrosterone sulfate, progesterone, estradiol, cortisol), recommends using an immunoassay based on enhanced chemiluminescence [17]. Arguing for the practical application of the luminescent immunoassay (LIA) method, I.V. Evstigneev points out that “LIA allows one to determine with high accuracy and specificity the level of fT (free testosterone) in saliva, which is an adequate marker for assessing androgen status in men” [17]. The author, in his studies on determining the level of free testosterone in saliva, used special containers (SaliCaps, IBL – Hamburg, Germany) and a special tube connected to the container, which was made of a material that does not absorb steroids, to obtain and store saliva samples [17]. Therefore, having extensive practical experience in conducting saliva diagnostics, I.V. Evstegneev (2014) makes the following practical conclusion: “The level of free biologically active testosterone in saliva strongly correlates with the biologically active form of the hormone in the blood (cT plus albumin-bound testosterone).
The level of testosterone in saliva is a more adequate indicator of biological activity than serum testosterone, especially when the binding ability of specific transport globulin changes” [17]. When determining the level of cortisol in saliva, the author points out that “Determination of cortisol in saliva is used as an alternative method when conducting functional tests with dexamethasone, adrenocorticotropic hormone (ACTH), insulin, while the stressful effect of repeated blood sampling on the hypothalamic-pituitary-adrenal system is reduced (GGAS). Studying the concentration of free cortisol in saliva is more suitable for assessing physiological fluctuations in HPA axis activity over time compared to determining the level of cortisol in the blood” [17]. And it’s hard to disagree with this, especially having practical experience in conducting this kind of research both in clinical practice and among athletes. The author, when studying the determination of Free cortisol, used special saliva collection systems Salivette (Sarstedt) [7]. The resulting saliva sample was examined using the ECLA method on an automatic analyzer. After analysis, saliva samples were frozen at a temperature of – 70°C for ELISA using Salivary Cortisol ELISA Kit SLV-2930 (DRG) diagnostic kits [3,4,17].
The method proposed by LV. Belskoy et al., 2011б is quite well-reasoned and has proven itself positive over many years [1]. According to their opinion, based on many years of practical experience, “The most promising new diagnostic technology is the morphological study of biological fluids. The crystallographic (tesigraphic) method has significant sensitivity, and therefore has found wide application, first in the practice of forensic chemical analysis, and then in sports medicine. Data on microcrystallization of oral fluid can be used as a method for assessing the general condition of the human body and, in particular, to determine the state of the body before and after physical activity” [1]. To determine the type of saliva microcrystallization (MCS), the authors propose to use the methods of Leus P.A. and Puzikova O.Yu. [1]. It should be noted that this method has been used in practical medicine (gynecology, reproductive and forensic medicine) for quite a long time. Thus, the ability of saliva to crystallize with the “creation” of a pattern of varying degrees of severity, in different phases of the ovarian-menstrual cycle (OMC), is used in the practical application of natural family planning (NFP) methods, in particular in the work of the Arbor® minimicroscope and the Vesta device®”, as well as the “Maybe Mom®” test microscope for determining ovulation using a drop of saliva [4,21,22]. Crystallization patterns of saliva in different phases of OMC are presented in the Figure 1.
Figure 1
It should be noted that with a meager content of female sex steroids in the blood serum and saliva during the postmenstrual period of OMC (estrogens and progesterone), the degree of crystallization of saliva will be unexpressed, in the form of “sand”, as it approaches ovulation (preovulatory period of OMC) and, accordingly, the level of sex steroids in the blood and saliva increases, clusters of crystals appear in the saliva sample under a microscope. The peak of crystallization, with a pronounced manifestation of the process of their loss, in women occurs in the middle of their individual OMC, which is characterized by the maximum concentration of microcrystals both in the blood serum and in saliva samples [4,21,22]. It should be noted that similar changes occur in the cervical mucus of women, depending on the phases of its OMC, which was the basis for such a practical EPS method as the Ogino-Knaus method [4,21,22]. Informative and relevant, from a practical point of view, is to determine the level of cortisol and adrenal hormones in saliva. This will be very important and practically in demand among female athletes, with clinical manifestations of hyperandrogenism of varying degrees, especially in female athletes, with an identified shift in the values of the index of sexual dimorphism (SDI) in sexual somatotypes – from gynecomorphic to mesomorphic and, especially, andromorphic [3,4-17,6,16,20].
Since this dynamic phenomenon, with changes in the values of the Index of Sexual Dimorphism (SDI), is inherent in many female athletes in most types of modern sports, express diagnostics of the level of cortisol and a number of other hormones (hypothalamic-pituitary, steroid) in saliva will help in clarifying the course of the adaptive process changes and correction of reproductive health disorders in many athletes, both in reproductive age, and in young athletes in prepuberty and puberty, and former athletes in premenopause and menopause [3,4-17,6,16,20]. Also relevant for the phenomena of hyperandrogenism and the phenomena of hirsutism will be the determination in saliva of such an indicator as the level of androstenedione, which is formed in the adrenal glands and gonads and is an intermediate product in the synthesis of both testosterone and estrone [3,6,4-17,20]. According to T.P. Vavilova et al., in the saliva of women, the concentration of androstenedione is normally low, but with hirsutism it increases 2-6 times. In men, the level of this hormone in saliva does not differ from that in women [3].
The practical application of saliva diagnostics in sports is given attention by such authors as S.S. Mikhailov and E.V. Rosengart. Their joint research, the data of which was published in 2008-2012, has significant theoretical and practical interest in matters of sports physiology. Thus, in their joint review article “Saliva as an object of biochemical control in sports”, 2008 [7], provides a lot of valuable information about changes in the indicators of a number of hormones in saliva during physical activity of varying intensity in athletes [3]. The authors show the possibilities of saliva diagnostics in determining the level of acid-base balance (ABB) and immune status (in particular IgA values), amylase, peroxidase, etc. in athletes, as well as cyclic changes in the spectrum of steroid hormones in athletes in a number of sports (rugby, volleyball, handball, judo) [7,8,10]. An important point in carrying out saliva diagnostics of various directions, incl. and when determining the level of hormones in saliva, there is a unity of methods associated with collecting this biological material from patients, which, in our opinion, requires strict algorithmization. In this regard, in his many years of fundamental research concerning the determination of the level of a number of hormones in human saliva, T.P. Vavilova, I.G. Ostrovskaya, A.E. Medvedev (2014), give the following valuable practical recommendations: “Mixed saliva can be collected both in standard measuring glass containers and in special polypropylene containers, as well as using a moisture-absorbing cotton or synthetic swab placed at the bottom of the mouth. The reduced content of hormones in stimulated saliva samples may be due to their adsorption on paraffin and cotton swabs. Replacing a cotton swab with a synthetic one or using special systems such as Salivette (“Sarstedt”, Germany), Quantisal (“Immynalysis”, Canada), Saliva Collection System (“Greiner BioOne”, Austria) improves the quality of analysis and affects the level of hormones in studied saliva samples” [3].
This is a very valuable recommendation, which allows us to unify the algorithm and methodology for collecting saliva samples from a contingent of people (patients, athletes), in whose saliva it is planned to determine the level of the hormones being studied. Further, the authors of the study provide valuable practical advice: “The level of hormones in saliva is also affected by the conditions of its storage. A number of hormones, primarily steroid hormones (androgens and glucocorticoids), retain their properties when saliva samples are stored at room temperature for several days. The addition of preservatives significantly prolongs the stability of salivary steroid molecules, which is essential, for example, for sending saliva samples by mail” [3]. The authors focus special attention on the comparison of methods for determining hormones in saliva, their informativeness, advantages and disadvantages of different methods of saliva diagnostics [3]. In addition, the authors point to another, fundamentally important, in our opinion, problem. The point is that “the determination of various hormones is affected by the growing number of reagent kits on the world market from different manufacturing companies. In order to speak with complete confidence about the similarities or differences in the determination of hormone concentrations by one or another test system, it is necessary to eliminate possible variations that arise when conducting analyzes in different laboratories, in different cities, which is inevitably associated with freezing/thawing of samples, their transportation. Therefore, it is advisable to determine the concentrations of hormones in saliva within the same laboratory using test systems from the same manufacturer” [3].
Conclusion
1. Saliva diagnostics is a promising modern method for diagnosing the level of hormones and a number of other biologically active substances in the saliva of athletes, both at different stages of the training and competition cycle, and at rest.
2. As a simpler, non-invasive, accessible and accurate method, in most cases, saliva diagnostics is an alternative to studies with blood serum.
3. The use of saliva diagnostics in determining the level of hormones of the hypothalamic-pituitary, adrenal and ovarian zones in female athletes – diagnostics of their hormones in women, taking into account their individual characteristics and phases of OMC – a relevant and modern way of taking into account the adaptation of female athletes to physical activity.
4. It is necessary to standardize and algorithmize the saliva diagnostic procedure, with mandatory correlation of hormone indicators in blood serum and saliva.
5. The saliva diagnostic method in sports physiology and medicine should become an accessible and mandatory diagnostic method in any sports team when preparing athletes during training and competition.
A Porcine Study on Postoperative Pacing of the Gastro- Intestinal Tract
Introduction
Abdominal surgery results in paralysis of the gastrointestinal tract which can last from minutes to several days depending on the surgical trauma [1-4]. Especially major open abdominal surgery is prone to induce gastrointestinal paralysis and prolonged paralysis for more than 5 days can occur in up to 54% of patients undergoing open surgery for advanced abdominal cancer [1-3,5-8]. Prolonged paralysis results in a range of symptoms including abdominal distension, nausea, inability to tolerate enteral nutrition, and non-passage of flatus or stools. This is, furthermore, associated with increased morbidity, prolonged hospital stays and even increased mortality [9,10-12]. Unfortunately, treatment options are sparse and are essentially limited to naso-gastric suction, laxatives, and patience [3,9]. So far, it has not been possible to initiate gastrointestinal motility immediately postoperative. In heart surgery, postoperative dysrhythmias and bradycardias are treated by pacing through temporary pace electrodes placed during the operation [13]. The neuroelectrical anatomy of the heart and gastrointestinal tract, however, is in many ways build the same way [14,15]. Pacemaker centers in the heart – the sinus and sinoatrial node – equals in many ways network of Cajal cells, which lie in the myenteric region between the circular and longitudinal muscle layer and initiate electrical impulses commencing slow wave gastrointestinal contractions. These Cajal networks are located at several regions of the gastrointestinal tract. Other cells of Cajal are also distributed intramuscular along the muscle bundles of the gastrointestinal tract, and – like the Purkinje fibers in the heart-these cells make up a fast track conducting network which enables coordinated muscle contractions [11,16].
In spite of these similarities, pacing the gastrointestinal tract is only performed in a few selected diseases. Especially in diabetic gastroparesis and for faecal incontinentia different pacing methods are well-established treatment options [10,11,16]. Good results from gastrointestinal pacing have also been shown in colonic inertia using colonic pacing [12,17]. However, whether pacing can be used in gastrointestinal surgery to treat postoperative dysrythmia and paralysis, the same way it is used in heart surgery, is unclear. No contemporary research has been made in this area. In this feasibility study our objective is to initiate gastrointestinal contractions immediately postoperative using an external pacemaker as a novel treatment option of postoperative paralysis. Furthermore, we sought to assess optimal pace settings and locations of the gastrointestinal tract in a postoperative setting.
Materials and Methods
Study Design
The study was conducted as an acute porcine observational study. The experimental animals were anesthetized and submitted for open abdominal surgery for two hours. Immediately postoperative, experiments were conducted investigating the effect of postoperative pacing of the gastrointestinal tract at the following preselected locations: The stomach, the duodenum, the jejunum, the caecum, and the rectum. The effect of the following pacemaker settings was tested at each location: Amplitude, pulse width, and pulse rate.
Experimental Animals
The study comprised 11 Danish Landrace/Yorkshire pigs, with a bodyweight of 60 kg each. Following the experiments, the pigs were euthanized, using an intravenous injection of pentobarbital 400 mg/ml (0,1 ml/kg), during continued anesthesia. The sample size was determined to accomplish the required combinations of pacemaker settings. The primary outcome was occurrence of an evoked gastrointestinal muscle contraction at the paced location.
Experimental Procedure – Anesthesia
The pigs were initially sedated with an intramuscular injection of 0.5 mg/kg midazolam (Dormicum®; Roche) at the research farm. Upon arrival at the laboratory, the pigs were anesthetized with an intravenous injection of 0.5 mg/kg midazolam and 6 mg/kg ketamine (Ketalar®; Warner Lambert) through an ear vein. The animals were intubated and placed on continuous anesthesia with sevoflurane aiming at a mean alveolar concentration of 2-2.7% and with 10μg/kg/h fentanyl (Haldid®; Janssen-Lambert) for pain relief. Each animal was monitored continuously for blood pressure, urine production and electrocardiogram during the entire experiment, with intermittent assessment of arterial blood gas values [18].
Experimental Procedure – Surgery
All experimental animals were subjected to two hours of surgery. We began with a large midline laparotomy from the xiphoid to the os pubis. Immediately after laparotomy the neck was incised on the left side and the neck vessels isolated. The internal carotid artery was cannulated in order to monitor blood pressure. The external jugular vein was cannulated in order to secure a safe venous access. After neck vessel cannulation the animals were subjected to abdominal surgery during the remaining operation time with the following procedures: Cholecystectomy (n=8), full mobilization of the spiral colon (n=9), segmental resection of left lateral liver lobe (n=4), non-anatomical liver wedge resection (n=3), Parietal abdominal wall and retroperitoneal peritonectomy (n=5), mobilization of the caval vein at liver level (n=2), resection of the distal pancreas (n=2), unilateral nephrectomy (n=2), splenectomy (n=4), subtotal hysterectomy (n=4), resection of gastro-colic ligament including the gastro-epiploic artery (n=2), extraperitoneal mobilization of the bladder (n=2), Mattox maneuver (n=1), or Pringles maneuver (n=9). No bowel or stomach resection was performed, thus, keeping the gastrointestinal neural connections intact. The vagal nerve was also not damaged.
Experimental Procedure – Pacing
After two hours of surgery pace wires were sutured on to the surface of the gastro-intestinal tract at the following locations: The stomach, 2/3 distal on the greater curvature; the duodenum, between first and second part; the small bowel, approximately 20 cm from the duodenum; the caecum; and the rectum, just below the recto-sigmoid junction. At each pace site two pace wires were attached one centimeter apart. Following a strict schedule multiple pace settings were tested at each location during continued anesthesia and with the abdomen open. A location was paced for two minutes or till an evoked muscle contraction occurred at the pace site. This was evaluated visually by the investigator. We used an external pacemaker, Test Stimulator 3625 (Medtronic Inc., MN), for testing the following amplitudes: 0.5V, 5V, 8V, and 10V volts. We used another external pacemaker, DISA Stimulater Unit type 14 E 11 (DISA, Denmark), for testing amplitude levels, 15V and 20V. We, furthermore, tested the three different pulse widths: 50μs, 200μs and 450/500μs (depending on pacemaker, data were pooled) as well as six different pulse rates: 5pps, 10pps, 25pps, 50pps, 80pps, and 100/120pps (depending on pacemaker, data were pooled). For each of the gastrointestinal locations (stomach, duodenum, jejunum, caecum and rectum), tests were performed for all amplitude levels (Volt) and combined with all frequencies (Hz). All frequencies were, furthermore, combined with each pulse width (μs) summing up to a total of 540 tests (See Figure 1). The pace current was calculated at selected pace site by measuring the voltage drop of a 10 Ohm series resistor and applying ohms law.
Figure 1
Figure 2
Ethical Considerations
The experiments were conducted according to the guidelines and with approval from the Danish Inspectorate of Animal Experimentation under the Danish Ministry of Justice.
Statistics
Data are presented as actual numbers of positive responses and in percent. Data are compared using logistic regression with clustering allowing for intragroup correlation. A p-value <0.05 was defined as a statistically significant difference. Data were analyzed using Stata IC 15.1 statistical software (StataCorp LLC, Texas, USA).
Results
Amplitude
Amplitude levels were tested in the range of 0.5-20V. For the different pace locations, the optimal amplitude levels were as follows. The stomach reached a contraction rate of 72% at 20V (P=0.02). The duodenum had the highest contraction rate at 8-15V (78-94%). Maximum contraction rate was at 10V (P<0.001). The jejunum had the highest contraction rate at 8-10V (94-100%). The maximum contraction rate was at 8V (P=0.005). The caecum generally had a high contraction rate especially at 10 and 20V (94%) (P<0.001). The rectum had the highest contraction rate at 8-20V (89-100%). The maximum contraction rate was at 10V (P=0.001). Generally, the low amplitude levels of 0.5 and 5V exhibited the lowest contraction rate, however the caecum and rectum still responded acceptably at this level. See Table 1 for the respective electrical currents as well as Table 2 and Figure 2 for further details on amplitude.
Table 1: Electrical current measured at selected locations.
Table 2: Number of contractions and odd ratios for different amplitude levels. Data is given as actual number (percent) and as odds ratio (OR) with 95% confidence interval. A hyphen predicts perfect success or perfect failure. N=18 measurements for each amplitude level at each location in gastrointestinal tract.
Figure 3
Pulse Width
Pulse width levels were tested in the range of 50-500μs. For the different pace locations, the optimal pulse width levels were as follows: The stomach reached 69% contraction rate at 500μs (P=0.1). At lower pulse width levels, the contraction rate was considerably reduced (11-14%). The duodenum showed contraction rates between 50% and 64%. The maximum number of contractions was seen at a pulse width of 50μs (P=0.4). Likewise, the jejunum reached acceptable contraction rates at all pulse width levels (61-67%). The maximum number of contractions was seen at a pulse width of 50μs (P=0.4). This was even higher for both the caecum and rectum, which reached a high contraction rate for all pulse width levels of respectively 72-92% and 81-86%. The maximum number of contractions was seen at a pulse width of 200μs for both pace locations (P<0.001 and P=0.03 respectively). See Table 3 and Figure 3 for further details.
Table 3: Number of contractions for different pulse width levels. Data is given as actual number (percent) and as odds ratio (OR) with 95% confidence interval. N=36 measurements for each pulse width level at each location in gastrointestinal tract.
Pulse Rate
Pulse rate levels were tested in the range of 5-120Hz. For the different pace locations, the optimal pulse rate levels were as follows: The stomach contraction rate peaked at 39% at 120Hz (P=0.4). At lower pulse rate levels, the stomach did not respond well (22-28%). The duodenum exhibited the highest contraction rate at 25Hz (78%) (P=0.06). Generally, the duodenum had contraction rate levels above 50%. The jejunum also exhibited the highest contraction rate at 25Hz (72%) (P=0.2). Generally, the jejunum had contraction rate levels above 56%. The caecum generally had high contraction rates ranging between 67-89%. The highest contraction rate was seen at 10, 50 and 80Hz (P=0.006). This was even better for the rectum, which reached high contraction rates for all pulse range levels of 78-94%. The highest number of contractions was seen at 50Hz (P=0.03). See Table 4 and Figure 4 for further details.
Table 4: Number of contractions for different pulse rates. n=number of measurements per pulse rate value. Data is given as actual number (percent) and as odds ratio with 95% confidence interval. N=18 measurements for each pulse rate level at each location in gastrointestinal tract.
Figure 4
Discussion
In this study we have demonstrated the possibility of pacing the gastrointestinal tract in the postoperative setting. We, furthermore, identified both optimal settings of the pacemaker as well as possible pace locations.
From the beginning of heart surgery temporary pace electrodes were used to treat postoperative bradycardias. Inspired by this, Bilgutay et al. developed a gastrointestinal pacemaker in 1963 in order to treat postoperative paralysis. Although heart surgeons implanted temporary pacemakers directly in the muscle of the heart Bilgutay et al. chose to pace the gastrointestinal tract using nasogastric tube electrodes as this method is less invasive [7]. Initial experiments seemed promising, but further clinical studies conducted in the same period did not support the concept why this line of research was abandoned until now [9-14]. These clinical studies, however, were small – comprising 17-48 patients. Furthermore, the method of intraluminal ventricular pacing implies great risk of connection failure as electrodes are placed blindly inside the stomach. In a clinical setting with paralysis the stomach must be filled and dilated and as the electrodes are not attached at the stomach wall a secure connection must be difficult to obtain [9]. In order to secure a proper connection between the pace wires and the gastrointestinal tract another approach could, therefore, be to implant temporary pacemakers directly on the wall of the gastrointestinal tract [19]. This was the approach we used in the present study with promising results.
We were, thus, able to initiate local contractions in the gastrointestinal tract in a postoperative setting. Pacing the GI-tract is a technique used in other diseases as well. Especially in gastroparesis implantation of a permanent pacemaker is a well-established method and can also be performed as a minimally invasive procedure [20,21]. Also, sacral nerve stimulation for anal incontinence is an integrated method in clinical practice and has proven excellent results. The method does not entail direct stimulation of the bowel or sphincter but uses indirect stimulation through the sacral nerve [10]. The method of sacral nerve stimulation is also being tested for irritable bowel syndrome as well [11]. Shafik et al. also presented a method to treat constipation due to colonic inertia with promising results. They used a similar method as us with direct stimulation of the colon. They, however, used an endoscopic method to place the electrodes within the mucosa and muscularis [12,17]. Interstitial cells of Cajal have been described for the first time by Spanish neuroanatomist Santiago Ramon y Cajal (1852–1934) in 1889. They are pacemaker cells and are found throughout the gastrointestinal tract from the esophagus to the internal anal sphincter. They are organized in a network placed both intermuscular and intramuscular [14,22]. They initiate slow wave impulses starting at the antrum and major curvature of the ventricle and then distribute the impulses along the small bowel. In the colon the initiation of the electrical impulses is still debated.
However, experimental studies have identified at least four potential pacemaker sites located at the cecal pole, the caecocolonic junction, the mid-transverse colon, and the colosigmoid junction. At these sites groups of myenteric Cajal cells are located between the circular and longitudinal muscle layer [12,17]. Our choice of pacing location was defined from this knowledge and from an intention to include representative sites from the entire gastrointestinal tract. The stomach required an excitation level of 10-20V before a minimum number of contractions could be detected. This indicates that direct muscle pacing is the most effective approach for the stomach as opposed to nerve stimulation. As we chose to place our electrodes on the wall instead of into the wall of the gastrointestinal tract, higher amplitudes could be needed to penetrate the thick wall of the stomach. This might explain the difference when comparing clinical pace settings in treatment of gastric paresis where a lower amplitude is generally needed. In the clinical treatment of gastropareses electrodes are placed within the layer of muscularis propria [20,21]. Furthermore, clinical pacemakers typically do not reach higher levels than 10.5V. This will practically make clinical trials with higher amplitude levels impossible at the moment. Whether these high amplitude levels also entail a clinical side-effect such as stimulation of the muscles of the abdominal wall is also unanswered as we performed our studies at open abdomen conditions. In the other locations of the gastrointestinal tract contractions were possible for most of the amplitude levels except from 20V at the jejunum, where only few contractions were seen.
The efficacy for different pulse widths as well as pulse rates did not differ much in our study indication that both nerve and muscle may be stimulated postoperatively with the same result. According to the literature, a number of different settings have been utilized in different experiments at different segments of the gastrointestinal tract with positive outcome [23-25]. The exact pulse rate and width may, thus, not be that important for postoperative gastrointestinal pacing, as long as it is within the range of the current study. Only for the stomach we found a noticeably better effect in the high pulse rate as well as the high pulse width compared with the lower settings. This effect was also different from the settings used clinically for gastric pacing regarding the pulse rate [26]. Again, the extra-serosal placement of the electrodes along with the thick wall of the stomach may contribute to this result. However, when transforming our experiment to clinical use one must also bear in mind which location would be suitable clinically to place potential pace wires for postoperative pacing. Off course the stomach could be obvious as normal slow waves starts here. The stomach is also the only location that is used for direct muscle pacing in a clinical setting in the treatment of gastroparesis.
The fact that the stomach is adjacent to the abdominal wall diminishes the amount of wire inside the abdomen considerably. The wire inside the abdominal cavity entails a risk of obstructing ileus. The duodenum is not that obvious to use in a clinical setting as most of the duodenum is located retroperitoneally in humans. Likewise, the small bowel is not the best clinical pace site as the small bowel on the other hand is very mobile. Furthermore, the small bowel is not the starting point of the slow waves and are. Additionally, the small bowel is normally the first bowel segment to initiate contractions after surgery why pacing the small bowel seems less important. The colon and rectum is normally the last part of the gastrointestinal tract to initiate contractions after surgery and could, consequently, also be a good location to place pace electrodes [9]. The exact placement would also depend on what kind of surgery and bowel resection is performed (Figure 5).
Figure 5
Study Limitations
This was an acute study with many measurements performed on the same animal. Sometimes migrating motor complexes occurred and further measurements would have to be put on hold till these contractions ceased. Most often, however, we would register segmental contractions in the near vicinity of the pace electrodes only, and then we would move on to the next location. However, it seems likely that if we kept pacing more than the 2 minutes, the local segmental contractions would develop into migrating motor complexes. This must be verified in a chronic animal model before human studies can be carried out. Another limitation of our study is the porcine nature of our experiment. Ultimately, these results must be reproduced in a human model before expanding the results into general clinical practice. However, further animal studies are needed before initiating human studies.
Conclusion
In conclusion, we were able to initiate local gastrointestinal contractions immediately postoperative using an external pacemaker. We, furthermore, determined optimal pace settings for pacing the gastrointestinal tract. These results may serve as the first step towards a possible treatment option for postoperative paralysis.
Acknowledgements
The authors would like to thank the staff at the Department of Clinical Medicine, Aarhus University, especially animal technician Kira Graahede. We would, furthermore, like to thank the Biostatistical Advisory Service at Aarhus University for statistical assistance. The study could not have been performed without financial support from Arvid Nilssons Fond and Helga and Peter Kornings Fond to which we are thankful.
Financial Support
Arvid Nilssons Fond and Helga and Peter Kornings Fond.
Authorship Statement
• Jonas Amstrup Funder: planning and conducting the study, collecting, and interpreting data, and drafting the manuscript.
• Anne Kraushaar Martensen: planning and conducting the study, collecting, and interpreting data, and drafting the manuscript.
• Tommy Bechsgaard: conducting the study, collecting, and interpreting data, and drafting the manuscript.
Making the Case for Using Exploratory Structural Equation Modeling Within Biomedical Research
An Overview of Factor Analytic Techniques
Since its inception in the early years of the 20th century (Spearman [1,2]), factor analysis has been used extensively in applied research across numerous disciplines. The fundamental purpose of factor analysis is to establish the number and nature of latent variables or factors that explain associations among observed scores. A factor is an unobservable variable that affects more than one observed score and accounts for correlations among those scores. Common applications of factor analysis are to determine whether interrelationships among observed indicators can be accounted for by a smaller number of underlying latent constructs (Brown [3]), investigate convergent and discriminant validity for such indicators and constructs (Millon [4]; Van de Vijver & Leung [5]), serve as a method of data reduction (Cox, et al. [6]), and provide a mechanism for investigating and testing theoretical models (Matsunaga [7]). Over the years, Thurstone’s [8] “simple structure” common factor model has attracted the most attention. Within this model, each observed score or indicator is represented by one or more common factors and an error term. The variability of each indicator score is divided into two parts: common variance shared among indicators (i.e., communality) and unique variance that is specific to the indicator or due to random measurement error (i.e., uniqueness). The common factor model encompasses two primary types of factor analyses: Exploratory Factor Analysis (EFA) and Confirmatory Factor Analysis (CFA; Joreskog [9,10]). The goal of both approaches is to use a reduced number of distinguishable latent variables or factors to explain observed associations among the indicators examined (Brown [3,11]).
Exploratory Factor Analysis (EFA)
EFA is driven by data in the sense that it does not require that the number of factors or the pattern of relationships among latent factors and indicators be explicitly specified in advance. EFA is typically used in preliminary research to determine the number of common factors needed and identify which observed scores are the best indicators of the latent factors in relation to factor loadings that represent the relationships between observed scores and latent factors. The number of common factors can be determined based on a variety of methods (see, e.g., Brown [3,11]). Ideally, each observed score would be more EFA is driven by data in the sense that it does not require that the number of factors or the pattern of relationships among latent factors and indicators be explicitly specified in advance. EFA is typically used in preliminary research to determine the number of common factors needed and identify which observed scores are the best indicators of the latent factors in relation to factor loadings that represent the relationships between observed scores and latent factors. The number of common factors can be determined based on a variety of methods (see, e.g., Brown [3,11]). Ideally, each observed score would be more
Confirmatory Factor Analysis (CFA)
CFA is an extensively used structural equation method for testing theoretical models that represent relationships between observed scores and latent factors. In contrast to EFA, CFA is theory-driven and highly restrictive in that the researcher must specify the number of factors and how observed indicators relate to those factors. In most common applications of this method, researchers allow each indicator to load on one targeted latent factor but not on other factors to achieve the clearest simple structure. CFA is suitable for directly testing theoretical models and is more parsimonious than EFA (Brown [3,11]).
Exploratory Structural Equation Modeling (ESEM)
ESEM is a more recent approach to factor analysis (Asparouhov Muthén, et al. [12-15]) intended to overcome the highly restrictive nature of CFAs by allowing indicators to have non-zero (i.e., weak, or typically negligible) loadings on non-targeted factors, while still retaining the advantage of CFA in directly testing prespecified theoretical models. ESEM is more data-driven than CFA by allowing indicator scores to load on all factors but expecting those scores to load noticeably higher on targeted than on non-targeted factors. Recent studies have revealed that, when samples are of adequate size, ESEMs produce better model fits and more precise parameter estimates than do CFAs (Asparouhov & Muthén [12, 16-22]).
An Empirical Example
To illustrate advantages of ESEMs, we include selected results from a recent dissertation study by the first author (Hong [23]) in Table 1. The data reported represent responses from 447,500 residents in the United States (39% male, 61% female; mean age = 24.93), who completed the International Personality Item Pool NEO 120 questionnaire (IPIP-NEO-120) that we obtained from a publicly accessible university website (https://osf.io/tbmh5/) created to enhance research into personality-related constructs (Johnson [24]). The IPIP-NEO-120 has 120 items that measure the Big Five personality domain constructs: Agreeableness, Conscientiousness, Extraversion, Neuroticism, and Openness to Experience. Each domain scale has 24 items with six nested 4-item facet subscales (see the note to Table 1 for names of all facets within each domain). We conducted separate correlated multifactor CFA and ESEM analyses for each personality domain with factors corresponding to the six facets included within a given domain. In Table 1, we report three model fit statistics for each analysis. These include the Comparative Fit Index (CFI), Tucker-Lewis Index (TLI), and Root Mean Square Error of Approximation (RMSEA). In keeping with guidelines suggested by Hu and Bentler [25], we considered fits to be respectively acceptable and excellent with values of 0.90 and 0.95 or higher for CFIs and TLIs and values of 0.08 and 0.06 or lower for RMSEAs. As can be seen in Table 1, ESEMs provided noticeably better model fits than CFAs in all instances and matched or exceeded all criteria for excellent fits with only one exception (the TLI = 0.947 for Extraversion).
Table 1: Fit Statistics for CFAs and ESEMs and Average Off-Target Loadings for the ESEMs.
Note: Subscale facets for the personality domains described in the table include: Trust, Morality, Altruism, Cooperation, Modesty, and Sympathy for Agreeableness; Self-Efficacy, Orderliness, Dutifulness, Achievement-Striving, Self-Discipline, and Cautiousness for Conscientiousness; Friendliness, Gregariousness, Assertiveness, Activity Level, Excitement-Seeking, and Cheerfulness for Extraversion; Anxiety, Anger, Depression, Self-Consciousness, Immoderation, and Vulnerability for Neuroticism; and Imagination, Artistic Interests, Emotionality, Adventurousness, Intellect, and Liberalism for Openness to Experience. CFA: confirmatory factor analysis; ESEM: exploratory structural equation modeling; CFI: comparative fit index; TLI: Tucker–Lewis index; RMSEA: root mean square error of approximation. All analyses were based on correlated multifactor models using maximum likelihood parameter estimation. *Within the CFAs, all off-target loadings are set equal to zero.
For CFAs, in contrast, CFIs or TLIs never reached levels for excellent fits and failed to achieve acceptable fits across fit indices within three of the five personality domains (Agreeableness, Extraversion, and Openness to Experience). As would be desired, factor loadings for non-targeted factors in the ESEMs, on average, were negligible in size, ranging from 0.014 to 0.042 over the five personality domains.
Conclusion
Our intent in this brief article was to introduce readers to possible benefits of applying ESEMs within scientific research studies by combining the best aspects of EFAs and CFAs. To facilitate applications of ESEMs, routines for analyzing such models are now available in the computer packages Mplus (Muthén & Muthén [26]) and R (Prokofieva et al. [27]). Examples and computer code for analyzing the multifactor ESEMs described here as well as hierarchical and bifactor ESEMs can be found in Hong [23] and (Hong, et al. [28]. For more in-depth information about the nature and relative advantages of ESEMs over other procedures, we direct readers to the comprehensive treatment of such models in (Marsh, et al. [13]).
A Review on the Mechanism of Different Insecticide Resistance in German Cockroach (Blattella Germanica) in Worldwide
Background
Cockroaches have existed for 300 million years [1]. Among more than 4,600 known species of cockroaches globally, only thirty are considered household pests [2]. Among them, the Germanallergiesch, Blattella germanica (Figure 1) is a common domestic pest species of medical and economic importance [3] because they have high adaptability to human life and a wide range of habitats for survival [4] and transmits various pathogens Mechanically via feeding mechanisms, cast skins originating from the molting, feces and secretions and causes allergic [5]. So, the control of this important human home pest is necessary. Control German cockroaches as the important indoor sanitary pests in urban worldwide are increasingly hard due to their high adaptability, strong fecundity and extensive insecticide resistance [6]. The utility of chemical pesticides is still the most important tool for control of these cockroaches [7]. World Health Organization (WHO) lists insecticides used to control cockroaches [8]. A wide range of chemical insecticides are approved.
Figure 1
These are organophosphates (chlorpyrifos, chlorpyrifos-methyl, diazinon, fenitrothion, malathion, pirimiphosmethyl), neonicotinoids (dinotefuran, imidacloprid), carbamates (bendiocarb), hydrazine (hydramethylnon), arylpyrazole (fipronil) inorganic (boric acid), insect growth regulators (fenoxycarb, flufenoxuron, pyriproxyfen, hydroprene), pyrethroids (alpha-cypermethrin, beta-cyfluthrin, bifenthrin, cyfluthrin, cyphenothrin, d,d-trans-cyphenothrin, cypermethrin, deltamethrin, esfenvalerate, etofenprox, lambda-cyhalothrin, permethrin), and sulfonamide (sulfluramid). Unfortunately, use of approved insecticides are limited due to resistance to these products [9]. Insecticide Resistance Action Committee (IRAC) defines resistance to toxins as follows [10] ‘a heritable change in the sensitivity of a pest population that is reflected in the repeated failure of a product to achieve the expected level of control when used according to the label recommendation for that pest species. Frequency in response to insecticide selection pressure increases because alleles confer appropriate resistance factors. As a result, a population can survive the lethal concentration of an insecticide that normally kills a wild population [11].
Resistance Mechanisms
Behavioral resistance, target site insensitivity, reduced cuticular penetration and metabolic detoxification are mechanisms of insecticide resistance in German cockroaches [12].
Monitoring Techniques
The most widely used bioassay methods on the susceptibility of cockroaches to insecticides are surface contact bioassays (jar test), topical application and toxic baits [13]. In topical applications, a specific amount of insecticide is placed on either the ventral thorax or abdomen of insects. The jar test that accepted worldwide standard method for monitoring resistance in the German cockroach depends on tarsal contact with deposits of insecticide in a glass jar. these methods were tested with numerous insecticides: diazinon, bendiocarb; chlorpyrifos; propoxur, cypermethrin and propoxur, chlorpyrifos, fenitrothion, pyrethrins, and cypermethrin and detected that regardless of the insecticide, jar tests are more practical because they rely on tarsal contact with insecticide deposits, but compared with the more precise topical applications, not sufficiently sensitive because they always produce smaller resistance ratios.
Insecticide Resistance in German Cockroaches
The present research desirable to develop database and monitoring strategies to estimate the susceptibility of German cockroach populations in worldwide with the aim to advance judicious use of insecticides in cockroach control program. Most cockroach susceptibility tests worldwide are performed on Blattella germanica, the results of which are classified into groups of insecticides and are presented. After the housefly, the German cockroach is the second most insecticide resistant urban pest, reported to be resistant to 45 insecticides [9]. Heretofore, more than 282 cases resistance are observed worldwide that first resistance was reported with Organochlorines (chlordane) in Texas in 1952 [14], in 1964 to organophosphates, in 1968 to carbamates, in mid-1980s for pyrethroids, in 1992 to sulfluramid, and in 1994 to abamectin. Among forty-five field-collected strains of German cockroaches examined for resistance to insecticides by the time-mortality response method, only low to moderate resistance to chlorpyrifos, and acephate was detected. strains showed high Resistance to malathion, carbamates, propoxur and bendiocarb and detected resistance to pyrethrins in half of the strains. High resistance was uncommon with propoxur. in some of the strains was detected resistance to the pyrethroids allethrin, permethrin, phenothrin, fenvalerate, and cyfluthrin [15].
Among seven fields collected population of German cockroaches from Tehran City, five showed high resistance to organochlorine DDT indicating the possible cross resistance between three pyrethroid insecticides permethrin, cypermethrin and cyfluthrin and the organochlorine DDT [16]. Pyrethroids are synthetic derivates of pyrethrins, which extract of Chrysanthemum cinerariaefolium flower. These pyrethroids are now used in many synthetic insecticides and are highly effective and efficient neurotoxic insecticides against insects with mode of action on the voltage-sensitive sodium channels [17]. Due to the low efficacy and mammalian toxicity of pyrethroid insecticides, these compounds have become very popular for the control of German cockroaches, but in some populations has been documented control failures due to the development of resistance in many parts of the world [18,19]. Blattella germanica has shown Resistance level till now about up to 468.00× against pyrethroids (beta-cyfluthrin, deltamethrin), 62.50× against the carbamates (propoxur), up to 28.80× against OPs (chlorpyrifos), and 10.0× against phenyl pyrazole (fipronil). As well as moderate level of resistance is found in cockroaches against neonicotinoids including imidacloprid (0.8–3.8×) and the oxadiazines like indoxacarb (1.4–5.3×) [20].
At the study used of 2 German cockroaches’ strain and the H strain was more resistant to all of the three carbamate insecticides than the D strain, that the order of resistance for H strain was carbaryl > propoxur > bendiocarb [21]. Resistance to the carbamate insecticides in several strains of B. germanica have previously been reported in the Britain, United States, Malaysia, Cuba, India, China, Singapore, Taiwan, South Korea, and Iran [21]. In a project investigated the resistance development of the German cockroach to Chlorpyrifos for 23 generations from the 5th instar nymphs of susceptible cockroaches, using Chlorpyrifos 50% lethal dose (LD50) as the insecticide selection pressure by topical application method. Obtained a highly resistant German cockroach cohort to Chlorpyrifos, which the resistance ratio was 21.63, after 23 generations of selection from susceptible strain cockroaches [22]. Two strains of German cockroach (Baygon-R and Pyr-R) showed 4-fold cross-resistance to imidacloprid that even do not be suppressed by PBO [23]. The Munsyana strain of German cockroach collected in Indana and was found to have high-level resistance to fenvalerate, displaying 825-fold levels of resistance by topical application [24].
A German cockroach, Blattella germanica (L.), strain Cincy was collected in Cincinnati, OH that gel bait-resistant and showed a high level of behavioral resistance to Avert (0.05% abamectin) and Maxforce FC (0.01% fipronil) gel baits. Topical application assays demonstrated moderate levels of physiological resistance to abamectin and fipronil with resistance ratios (based on LD50 values from topical applications) 2.5 and 8.7, respectively [25]. A German cockroach showed level of resistance to permethrin and deltamethrin 97 and 480-fold respectively compared with a susceptible strain [26]. German cockroaches collected from a number of field locations, ranging from Florida to South Korea, exhibitioned an avoidance behavior to a bait formulation. Cockroaches collected from locations that exposed with this formulation and had a history of treatment illustrated bait avoidance, whiles field and laboratory strains with no prior exposure ingested and were susceptible to the toxic bait. so, behavioral resistance had developed in these insects. Selection experiments showed that several susceptible strains were potentially capable of developing behavioral resistance [27]. populations of the Blattella germanica collected from various localities in peninsular Malaysia were tested for their susceptibility to commonly used insecticides.
The result showed Low to high levels of resistance to carbamates (1.8 – 65.2X) while resistance to organophosphates was low (1.1 – 4.3X). One strain exhibited high resistance to malathion (>275X). Eleven strains tested showed low to high resistance to DDT (1.3 – 40.7X). Resistance to pyrethroids ranged from 1.1 – 17.6X [28]. A strain of Blattella germanica with highly resistant to DDT has been reported from Europe [29]. Some populations of German cockroach were collected for testing susceptibility to several different insecticides by a topical application method in compared to an insecticide-susceptible strain. Extremely high to high levels of resistance in bifenthrin, deltamethrin, fenvalerate and low to moderate levels of resistance were observed in cypermethrin, permethrin, chlorpyrifos, chlorpyrifos-methyl and fenthion [30]. The three strains of the German cockroach observed different levels of resistance to permethrin and cypermethrin and low to moderate levels of bendiocarb resistance and low level of chlorpyrifos resistance based on resistance ratios compared with susceptible strain [31]. Collected populations of B. germanica from different localities in Taiwan island were evaluated for their resistance to deltamethrin, propoxur, and fipronil using the surface-contact method. Results had indicated that resistance exist and high deltamethrin resistance in some strains could affect in reducing effectiveness of indoxacarb, fipronil and imidacloprid baits [32].
The German cockroaches were provided from Sanandaj in Iran and evaluated with Surface contact method for bioassay using standard glass jar procedure. The results indicated that the German cockroach was resistant to malathion and propoxur while susceptible to lambda-cyhalothrin [7]. DDT has a long residual action of more than 6 months and is relatively economical, similar to pyrethroids. However, existence of high frequency of mutations in the knockdown resistance gene (kdr), cause conferring cross-resistance to DDT and pyrethroid insecticides; as well as several countries reluctant to register or utilize DDT due to perceived environmental or export concerns [33]. The German beetle (Blatella germanica (L)) strain, Apyr-R, was collected from Opelika, Alabama after control failure with pyrethroid insecticides and the levels of resistance to permethrin and deltamethrin were 97 and 480 times higher than the susceptible strain, respectively. Incomplete suppression of pyrethroid resistance with DEF and PBO shows that one or more mechanisms have rol in resistance. cuticular penetration is one of the barriers for the efficacy of pyrethroids against German cockroaches [26].
Survey the effect of exposure to baits containing fipronil, indoxacarb, or hydramethylnon on the development of physiological resistance to the same and other insecticides in a number of German cockroach strains indicate that long exposure to baits fipronil or indoxacarb developed physiological resistance to these compounds but no increase was detected in response to hydramethylnon bait. Furthermore, exposure to fipronil bait caused increased cross-resistance to indoxacarb and exposure to indoxacarb bait did not increase cross-resistance to fipronil. Neither fipronil nor indoxacarb bait exposure enhancement resistance to hydramethylnon. exposure to toxic baits, particularly fipronil, have an important role in the expansion of insecticide resistance, including cross-resistance, in German cockroaches [34,35] (Table 1). This review shows studies conducted have great different test in the using insecticides, concentrations methods and formulations. The notable point is that a considerable population of German cockroaches have become resistant to large numbers of insecticides from different groups in over of world. Multiple physiological-based mechanisms including reduced penetration by thickening or remodeling cuticle, metabolic resistance and target site insensitivity (kdr mutations) are associated with Blattella germanica insecticide resistance.
Table 1: Reports of resistance to insecticides in German cockroach populations [35].
Despite the spread of resistance in this pest, various application methods such as sprays and baits using different modes of action classes rather than insecticides from within the same IRAC mode of action group should be applied in a rotational strategy to provide control and delay the rapid extension of resistance in all species. The use of insecticides is an important element of health and urban pests’ management. Resistance to insecticides and the significant shortage of pesticides suitable for control is a serious threat that requires special and immediate attention from health authorities, researchers and the pesticide industry. Insecticide resistance management should be an integral part of pests control programs, and the structure, human resources, and other resources required should be given high priority.
Comparison of Venlafaxine with Other Antidepressants for the Treatment of Major Depressive Disorder: A Narrative Review
Introduction
Depression is the leading cause of disability worldwide. The number of people living with depression increased by around 18% between 2005 and 2015, and it is estimated that depression affects 322 million people, or about 4% of the world’s population [1]. Pharmacotherapy and psychotherapy are the two mainstays of depression treatment. In particular, second-generation antidepressants, including selective serotonin reuptake inhibitors (SSRIs), are the first-line options in the pharmacological management of major depression [2]. However, meta-analyses have shown that approximately only 50% of patients under psychological treatment seem to achieve normal functioning, with cognitive, interpersonal psychotherapy being the most effective approaches [3]. Tricyclic antidepressants (TCA) and monoamine oxidase inhibitors (MAOIs) like amitriptyline, imipramine, desipramine, isocarboxazid or phenelzine tranylcypromine have been the only available antidepressants for a long time. Despite compounds of these both classes have proven to be effective, adverse events, the use of suboptimal doses and potential drug-drug and drug-food interactions have arisen as caveats linked to these medications [4,5]. TCAs have shown non-specific serotonergic and noradrenergic activity, as well as a potential non-selectivity towards muscarinic cholinergic, α1-adrenergic and H1 histaminergic receptors, which might result in dry mouth, dizziness, blurred vision, constipation, sedation, and orthostatic hypotension and even death under overdose conditions [6-8].
In the case of MAOIs they also bind to multiple receptors and can interact with tyramine causing potentially fatal hypertension, in addition to the appearance of adverse effects such as hypotension, bodyweight gain and sexual dysfunction [6]. These factors have limited their use in patients with major depression. The introduction of SSRIs and afterwards serotonin-norepinephrine reuptake inhibitors (SNRI) was a major advance in the management of depression. Although SSRIs could have become the first-line treatment for several psychiatric diseases like uncomplicated unipolar depression and dysthymia, their effectiveness/safety balance in major depression has been questioned [9]. This narrative review examines the effectiveness of venlafaxine, as first developed SNRI, in the management of major depressive disorder, compared to SSRIs and other dual-acting antidepressants of its pharmacological class.
Material and Methods
This article is a narrative review which covers the mechanism of action of venlafaxine and a comparison in efficacy with serotonin reuptake inhibitors and other dual-acting antidepressants. A literature search was performed in PubMed based on the keyword’s “venlafaxine”, “comparative study”, “SSRI”, “serotonin reuptake inhibitors”, “dual-acting antidepressants” and “major depressive disorder”.
Results
Mechanism of Action
Venlafaxine, or 1-[2-(dimethylamino)-1-(4-methoxyphenyl)-ethyl]cyclohexanol hydrochloride, is a bicyclic phenylethylamine derivative that, along with its major active metabolite O-desmethylvenlafaxine, inhibits presynaptic reuptake of serotonin (5-hydroxytryptamine; 5-HT), noradrenaline (norepinephrine; NA) and, to a lesser extent, dopamine (DA). Compared to other antidepressants, it interacts with more than one receptor site, since it affects both serotonin and norepinephrine receptors. However, it does not interact with α1-adrenergic, muscarinic cholinergic, H1 histaminergic, benzodiazepine or opioid receptors and does not inhibit monoamine oxidase. Therefore, it avoids adverse events like dry mouth, hypotension, and sedation. However, venlafaxine can interact with dopamine receptors, inhibiting its reuptake [6,9]. Therapeutic efficacy of venlafaxine lies on the fact that both serotonergic, noradrenergic and dopaminergic systems take part in the pathophysiology of major depression, thus the blockade of these uptakes might benefit patients with this major depressive disorder. Moreover, venlafaxine has demonstrated that at low doses acts as SSRI, whereas if it is used at higher doses, it acts as dual 5-HT and also NA reuptake inhibitor [6,10], conferring this drug a remarkable versatility in relation with this special antidepressant mechanism of action. The clinically significant noradrenergic effect of venlafaxine occurs fundamentally at the level of the central nervous system, and, therefore, venlafaxine has been proven safe at cardiovascular level in several studies. (Mbaya, et al. [11]) reported that venlafaxine, even at high doses above 300 mg/day (mean 346.15 mg; range 225–525 mg) did not have any clinical or statistically significant in PR, QT, QRSD and QTc interval values, neither tachycardia. More recently, (Behlke et al. [12]), in a secondary analysis of the IRL-Gray clinical trial, observed that venlafaxine did not significantly affect cardiac conduction in 169 adults older than 60 years depressed patients treated with doses up to 300 mg daily, since it did not prolong QTc or other electrocardiogram parameters, regardless of the serum concentration of this medication.
Superiority of Venlafaxine towards SSRIs as a First-Line Treatment
It is widely accepted that 5-HT, NA, and DA play an important role in mood regulation. SSRI antidepressants act selectively on serotonergic systems by blocking serotonin uptake pumps. Therefore, serotonin levels available in the space between neurons are increased, extending their effect in the brain, and thus improving mood in patients [13]. Although SSRIs are usually the first-line therapies in the pharmacological management of major depression, venlafaxine has proven to be more effective in the treatment of this condition [2]. In a randomized, double-blind trial which compared venlafaxine and fluoxetine in 382 outpatients with major depression, patients were randomly assigned to receive either medication, with the option of increasing the doses three weeks after the start of the trial if a poor response was achieved. Initially, both venlafaxine and fluoxetine reduced significantly mean Hamilton Rating Scale for Depression (HAM-D), Montgomery-Asberg Depression Rating Scale (MADRS) and Clinical Global Impression (CGI) scores, although no significant differences were observed between groups. However, after three weeks, higher CGI-I scores (Clinical Global Impression-Improvement score of 1 or very much improved) were observed among those patients who increased their venlafaxine doses compared to fluoxetine (p<0.05) [14]. In a comparison study with sertraline in the treatment of major depressive disorder through an eight-week, double-blind, randomized trial, statistically significant higher response rates were observed in the venlafaxine group (83% vs 68%, p=0.05).
In addition, a higher number of patients reported a HAM-D score lower than 10 in the patients in which venlafaxine was prescribed (68% vs 45%, p=0.008). Regarding remission rates, percentages stayed at 67% and 36% for venlafaxine and sertraline respectively (p<0.05) [15]. In an analysis performed across eight double-blind randomized clinical trials that compared venlafaxine and SSRIs among 2,045 patients with major depression disorders remission rates were 45% for venlafaxine, 35% for SSRIs and 25% for placebo (p<0.001 for all comparisons). In addition, effectiveness of venlafaxine was statistically higher than SSRIs and placebo from the second week onwards. Overall, a 50% greater chance of remission with venlafaxine therapy compared to SSRIs was observed (odds ratio [OR]=1.5, 95% CI: 1.3, 1.9) [16]. In a pooled analysis of 1,454 outpatients diagnosed with major depression across five double-blind, randomized studies that compared the efficacy of venlafaxine with fluoxetine for six weeks, both treatments demonstrated higher response rates compared to placebo. Superiority in response rates was observed with venlafaxine, compared to placebo, between weeks 3 and 6 (p<0.05). Additionally, higher remission rates were observed in the venlafaxine group from the second week to the sixth week, since the percentages of remitter patients were greater compared to fluoxetine (9% vs 5% at week 2 and 36% vs 28% at week 6, p=0.019 and p=0.003 respectively). Both treatments proved to be superior to placebo, although significant differences were observed from week 3 onwards for venlafaxine (18% vs 10%, p=0.003) and from week 4 onwards for fluoxetine (20% vs 14%, p=0.042), suggesting a faster effect of venlafaxine. Furthermore, venlafaxine was more effective compared to fluoxetine regarding psychic anxiety (14.0% vs 8.8%, p<0.05) and sense of guilt (16.9% vs 9.6%, p<0.05).
This superiority on anxiety symptoms, commonly associated to depressive symptoms in major depression, could explain the superiority of venlafaxine as antidepressant, versus fluoxetine [17] or even other SSRIs. Recently, in an eight-week, multicenter, randomized, single-blind, active-controlled trial among 184 postmenopausal women with major depressive disorder, a higher reduction in HAMD-24 anxiety/somatization factor scores was observed with venlafaxine compared to fluoxetine (least-squares mean difference -2.22, 95% CI: -7.08, -0.41, p=0.001). These results translated to a greater baseline-to-eight-week least-squares mean change of the anxiety/somatization factor scores (p<0.05 for all). Although both treatments achieved better results compared to placebo, authors concluded that venlafaxine led to an overall greater improvement in the treatment of postmenopausal major depression [18]. Regarding meta-analysis, (Einarson, et al. [19]) reported from 44 trials involving 4033 patients that venlafaxine had the highest mean success rate (73.7%) compared to SSRIs (61.1%) and tricyclic antidepressants or TCAs (57.9%). The statistical analysis showed that these differences were significant (p<0.001). Concerning risk/benefit balance, it is worth noting that the dropout rates due to adverse event and also due to lack of efficacy were numerically lower in favor of venlafaxine, in comparison with SSRIs and TCAs. (Nemeroff, et al. [20]) reported in a meta-analysis with 34 randomized double-blind studies in which remission rates in the treatment of depression were analyzed, that venlafaxine was statistically superior to SSRIs as a class. Remission rates of venlafaxine over different SSRIs were superior in 28 of the 34 studies, with remission rate differences ranging from -7% to 31%. Overall, venlafaxine treatment was associated with a 5.9% advantage over the SSRI class (95% CI: 0.038, 0.081).
In another meta-analysis by (Bauer, et al. [21]) including 63 clinical trials, an overall random effect OR of 1.15 (95% CI: 1.02, 1.29) across 29 studies was observed, which indicated that venlafaxine was significantly more effective compared to SSRI. In addition, a greater effectiveness in remission rates in the venlafaxine group was also reported, with random effects OR of 1.19 (95% CI: 1.06, 1.34). Finally, in another meta-analysis which included 26 randomized, double-blind clinical trials, a greater response rate with venlafaxine compared to SSRIs was reported, with an overall OR of 1.17 (range 0.65-2.78, 95% CI: 1.03, 1.34, p=0.02). Regarding remission rate, it also proved to be superior in those patients who received venlafaxine in comparison with SSRIs, with a mean OR of 1.13 (range 0.27-2.62, 95% CI: 1.0, 1.28, p=0.05). Individual comparisons reported that venlafaxine was relevantly superior towards fluoxetine regarding response rates (OR=1.28, 95% CI: 1.05, 1.55, p=0.01) [22]. In conclusion, as per our review, venlafaxine seems to be superior to SSRIs in terms of efficacy in first-line treatment of major depression. As a compilation of all the above, the results obtained in the clinical trials are summarized in Table 1 [14-18]; and the ones from the meta-analyses in Table 2 [19-22].
Table 1: Summary of clinical trials comparing venlafaxine towards SSRIs as first-line treatment.
Table 2: Summary of meta-analyses comparing venlafaxine towards SSRIs as first-line treatment.
Superiority of Venlafaxine Compared to SSRIs in Refractory Patients to First-Line Treatment
Disease remission is the main treatment clinical aim of major depressive disorder. However, estimations indicate that only about 50% of these patients respond to the treatment of SSRI, with around 30% of them only achieving response or partial remission [23-25]. Since switching to a second SSRI in refractory patients has proven to achieve from variable to any response [26-30], a clinical strategy often employed is prescribing patients with an antidepressant with a different mechanism of action. In such a scenario, venlafaxine might represent an interesting option for these refractory patients according to the data available in the scientific literature. In a multicenter, randomized, double-blind, double-dummy, dose-ranging, parallel-group trial that compared extended release venlafaxine with citalopram among 406 patients with depression who had been unresponsive to eight weeks of monotherapy with an adequate regimen of SSRI other than citalopram, a non-significant difference in the mean change from baseline to the final on-therapy evaluation on the HAM-D21 total score was observed (-17.0 for venlafaxine vs -16.5 for citalopram, p=0.4778) for the overall population. However, when analyzing the clinically relevant subset of patients who had a CGI-I score of 1 (very much improved) after 12 weeks, statistically significant superiority was observed in the venlafaxine group (p=0.024). Sub-analyses of the HAM-D21 scores showed that among the patients who were more severely affected by reporting baseline scores greater than 31, venlafaxine group was significantly superior in comparison with citalopram group on the estimated longitudinal change in the total score from baseline (least squares mean of 14.25 vs 17.78; p=0.0121).
This difference was also observed in the CGI-S (Clinical Global Impression-Severity) scores (1.94 vs 1.53, p=0.0359) [31]. Finally, in a five-year retrospective analysis which compared switching to venlafaxine or other SSRI after treatment failure with a SSRI, it was observed that the rate of patients achieving a CGI-S score of 2 or less (1 = normal, not at all ill; or 2 = borderline mentally ill) after the different treatments was statistically higher in the venlafaxine group (68.1% vs 58.9%, p=0.02) [32]. To summarize, and in view of the reviewed evidence, venlafaxine could be more effective than SSRIs also for the treatment of refractory to first-line patients diagnosed of major depression.
Superiority of Venlafaxine Towards Other Dual-Acting Antidepressants
Desvenlafaxine is the main active metabolite of venlafaxine. It has proven to inhibit neuronal uptake of both serotonin and norepinephrine and, to a lesser degree, dopamine [33,34]. Several placebo-comparative clinical trials have established the efficacy of this compound for major depressive disorder [35-37]. However, there is no evidence that its effectiveness is superior to that observed in other antidepressant therapies [38,39]. In comparison with venlafaxine, in a post-hoc pooled analysis of two double-blind, multicenter, placebo-controlled, parallel-group, venlafaxine extended release-referenced trials, venlafaxine showed overall better results than desvenlafaxine. After an eight-week treatment, the change from baseline of HAM-D17 scores were -14.26 and -14.56 for the 75-150 mg/day and 150-225 mg/day doses of venlafaxine, respectively; -14.21 for desvenlafaxine (200-400 mg/day); and -11.87 for placebo with both treatments showing statistically significant differences towards placebo (p<0.001). However, venlafaxine, especially at the 150-225 mg/day dose, showed a more rapid separation from placebo in terms of HAM-D17 score temporary change, suggesting a faster onset of the antidepressant effect.
Interestingly, statistical and -from our point of view- also clinically significant differences were observed concerning both doses of venlafaxine towards placebo regarding response rates (64% for 75-150 mg/day and 57% for 150-225 mg/day, p=0.033 and p=0.017 respectively) and remission rates for the patients that received the 150-225 mg/day dose (36%, p=0.003). Such venlafaxine differences in comparison with placebo were not observed in favor of desvenlafaxine, neither in terms of response nor in remission rates, suggesting a weaker antidepressant effect of desvenlafaxine, in comparison with venlafaxine [39]. It is worth noting that the 200-400 mg/day dose range of desvenlafaxine in the previous pooled analysis is much higher than the authorized dose range for desvenlafaxine in Spain (50-200 mg/day) [40]. Relevantly, and regarding regulatory issues, the proprietary company decided to withdraw desvenlafaxine through the centralized process of marketing authorization approval via EMA (European Medicines Agency) due to, during the assessment procedure, EMA expressed concerns and held a provisional opinion that the drug could not be approved for the treatment of major depressive disorder [41]. As a result, at this moment desvenlafaxine is marketed and available for prescription in only 3 European countries through national authorization procedures, including Spain [42].
Discussion
Individuals diagnosed with major depression disorder are usually difficult to treat, so an effective management of these patients as early as possible is crucial to achieve the best possible outcomes. Although narrative reviews have inherent limitations due to their design (mainly inclusion of heterogenous studies, each of them with a different design and study protocols, and selection bias of the reviewers), most studies addressed in this narrative review included large groups of patients and, therefore, the results have the sufficient statistical power to detect clinically meaningful differences. From our analysis, we have observed a clear trend of venlafaxine superiority in different treatment scenarios or clinical research methodologies, which, in our view, could help to overcome the mentioned methodology limitations. Results included in this narrative review point towards the fact that venlafaxine treatment is significantly more effective than SSRIs in the treatment of first major depressive episodes with no major adverse effects added. Furthermore, it showed some benefits against some other dual-acting antidepressants such as desvenlafaxine in the management of depression by triggering earlier and superior responses in patients. In addition, the benefits of venlafaxine against SSRIs in refractory patients, which pose an exceptional challenge to specialists, is of special clinical meaningfulness. In the clinical management of major depression, the clinical success of the first line treatment is very important since it avoids therapeutic failures and medication changes, including washout periods which can lengthen periods of treatment without clinical effectiveness and, hence, jeopardize adherence and increase the burden of illness. Furthermore, the proven and robust anxiolytic effect of venlafaxine is an added value in the treatment of major depression. All of the above, as per our review of its risk/benefit balance, venlafaxine can be considered as option of choice for the treatment of major depression disorder. Finally, in order to reduce pharmacological burden, help to ensure adherence to treatment, and facilitate progressive dose titration, extended-release oral pharmaceutical forms of venlafaxine which can allow a once-a-day posology including a wide range of different therapeutic dosages should be preferred.
Conclusion
This narrative review indicates that venlafaxine could present a better efficacy profile for the treatment of major depressive disorder, not only compared to treatments based on SSRIs, but also to some other dual-acting antidepressants such as desvenlafaxine.
Persistent Chyle Leak Post Mastectomy with Axillary Clearance: An Experience with Conservative Treatment vs Surgical Management
Background
Chylous fistula, a complication related to thoracic duct injury or its branches, may develop following neck or thoracic abdominal surgery. Chyle is a milky fluid made up of emulsified fats (long chain triglycerides) and lymph derived from the absorption of fats from the small intestine into the lymphatic system. However, chyle leak after mastectomy with axillary clearance is an unusual phenomenon. Seroma formation and chronic lymphoedema of the upper limb are far more common. The reported incidence of chylous fistula after breast cancer surgery is less than 0.5%. It may delay wound healing, extend hospitalization, impair the immune system, and affect the initiation of adjuvant therapy. Although rare, it is important that all breast surgeons should be aware that a chylous fistula can develop after axillary dissection and its available treatment options. As this is a rare complication there is a paucity of evidence in its management strategies. The aim in this systematic review is to conduct a qualitative and/or quantitative synthesis of the literature and provide more robust recommendations for its management (conservative vs surgical modalities) to minimize the gap of knowledge though this study [1-5].
Study Aim and Objectives
• To investigate the treatment of post operative chyle leak after mastectomy.
• To study the risk factors for the occurrence of post operative chyle leak in patients that underwent mastectomy.
• To evaluate the permissible length of conservative treatment before progressing to surgical options
• To compare the numerous proposed conservative and surgical options to stop chyle leak.
Patient
Patients that undergone mastectomy and developed chyle leak post operatively
Intervention
The feasibility of treating patients conservatively.
Comparison
To progress to surgical management once conservative treatment fails.
Outcome
To study the complete resolution of chyle leak with the available treatment options (complications, prognosis).
Methods
Searches
Embase, MEDLINE and Web of Science were searched up to 01/11/22 for articles published in English.
Embase: (‘chyle leak’:ab, ti OR ‘chyle leaks’:ab, ti OR ‘chyle leakage’:ti OR ‘chylothorax’:ti OR ‘thoracic duct’:ti) AND (‘mastectomy’:ab, ti OR ‘nipple sparing surgery with axillary clearance’:ab
MEDLINE (PubMed): ((chyle leak[title/abstract]) OR chyle leaks[title/abstract]) OR (chyle leakage[title]) OR (chylothorax[title]) OR (thoracic duct[title])) AND ((mastectomy[title/abstract])
Web of Science: TS=(“mastectomy” OR “chyle leak post mastectomy” OR “breast resection” OR “chyle leak post mastectomy”)
Case reports were examined for procedure type, duration of leak and treatment strategy. Nevertheless, we will contact the corresponding author, by e-mail, for missing data that is needed for the systematic review. The references of systematic reviews that come up in the search will also be searched manually [5-10].
Exclusion Criteria: case reports, case series (<20 patients), systematic reviews, editorials, conference abstracts, studies where full text not in English, studies that were not specific to chyle leaks after mastectomy. The trend in the management of chyle leak following mastectomy with axillary clearance are identified as follows:
1. Primary outcome: chylous leakage
2. Secondary Outcome:
Side affected.
Type of surgery.
Level of axillary clearance – level 1-3.
Age
Drain output – mls in first 24hrs, then average mls/24hrs overstay.
Management
Complications from procedure
Mortality rate
Measures of Effect
Primary outcome was the chyle leak rate, defined as the presence of milky, non-infectious discharge through the abdominal drain with triglyceride levels higher than 1.3 mmol/L and lower cholesterol levels than blood, was defined as chylous ascites.
Data Analysis
The data will be reviewed independently by two reviewers and demographic characteristics of the data set will be assessed. The level of axillary clearance, the status of the lymph nodes taken (metastatic or benign) and the number sampled, the diagnostic tool used, the date any chylous leak noted, initial management, chylous output, secondary management, and time of resolution will be assessed. Comparison with separate groups of those that need secondary surgical management and with those who has been treated successfully conservatively.
Data Synthesis
Risk ratio (RR) of dichotomous outcomes (i.e., chyle leak, overall morbidity, and anastomotic leak) and mean difference (MD) along with their variances of continuous outcomes will be studied. The heterogeneity will be estimated using the Cochrane’s Q test and I² statistic and explored by using meta-regression. Publication bias will be assessed by Funnel plot and Egger test. A two-sided P-value < 0.05 considered to indicate significance.
Data Extraction
Data relating to multiple outcomes will then be extracted from the studies selected for inclusion, pertaining to side, age, type of surgery, level of axillary clearance (if applicable), drain output, subsequent management, length of stay, complications, and mortality.
Risk of Bias Assessment
As the study revolves around case reports, most of the data collected will be subjective and anecdotal. Hence, risk of bias assessment will not be necessary, other than to recognize the limitations within the methodology.
Ethical Approval
No ethical approval will be subjected as systematic reviews do not involve human participants, collecting new data or conducting primary research.
Subgroup Analysis
A subgroup analysis of those that require surgical management and those that are managed conservatively may be conducted.
Timescale
My aim to complete the research should be in a time frame of 6 months (Dec 2023- May 2024). The breakdown of the tasks are as follows:
A. September 2023- December 2023
• Establish research question.
• Complete the research protocol.
• Formulate research strategy.
B. December 2023- March 2024
• Establish research design.
• To register the protocol with PROSPERO
• Scoping for data via many of the search tools
• Formulate and write up data analysis.
• Collaborate with other co reviewers.
C. March 2024- May 2024
• Establish the first and second draft of the systematic review.
• Proofread and to commit final checks to the dissertation.
• Write up the dissertation and submit the assignment.
Contingencies
There are a few constraints that could be anticipated from conducting this research which are.
• Lack of resources/literatures
• Time constraints
• Potential problem with programming tool (STATA, SPSS)
• Falling sick
• Financial constraints
• The alternatives that could be taken to minimize interruptions are.
• To prioritize the workflow of the research
• Identify electronic library resources that are needed.
• Identify support needed to access programming tools.
• Personnel that are unwell to stay home and to work remotely.
• Identify the research component which needs more time to be completed.
• Consider reaching out to funding agency program managers (Table 1).
Biomedical Journal of Scientific & Technical Research (BJSTR) 