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Changing Lives by Changing Minds: Reducing Cognitive Biases to Enhance Psychological Health

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DOI: 10.31038/PSYJ.2024654

Abstract

Our central thesis is that integrating multiple evidence-based techniques will offer the best results for optimizing psychological health. We believe that effectively addressing the escalating mental health crisis will require a comprehensive, multi-pronged, approach. Decades of research across the psychological and cognitive sciences have shown that errors in the way people think, called cognitive biases, have a profound impact on many aspects of psychological health. In this manuscript we review several research-based approaches for changing the way people think to improve psychological health. We review evidence on how different cognitive biases—negativity bias, framing bias, confirmation bias, and essentialism bias—impact various aspects of psychological health, and the benefits of mitigating those biases. We also review the important role of social perspective taking in psychological health and how cognitive biases, such as the curse of knowledge bias, the false consensus effect, the spotlight effect, and the fundamental attribution error, interfere with social perspective taking, further highlighting the merits of strategies to minimize these biases. In conclusion, we propose that an integrated, multi-pronged approach is the best way to address the unique and diverse challenges individuals face to maximize the benefits for individuals, and society as a whole.

Keywords

Psychological health, Cognitive bias, Social emotional health, Intervention, Education, Social perspective-taking, Mindset, Theory of mind, Anxiety, Depression, Coping

Introduction

The world is experiencing a mental health crisis! 1 in 8 people worldwide are struggling with their psychological health [1]. In the US, 1 in 5 adults currently have psychological health issues, placing a tremendous $300 billion burden on the economy in lost productivity and healthcare costs [2,3]. Frighteningly, the situation is getting worse. As just one example, the number of Canadians diagnosed with anxiety and depression has more than doubled in the last 2 decades and ERs have seen a 75% rise in psychological health-related visits, especially among younger individuals [3,4]. To prevent this crisis from escalating, there is an urgent need for more effective approaches to improve psychological health, not only for those currently struggling but also for the general public. Everyone can benefit from learning research-based strategies to enhance their psychological well-being. In this manuscript we review research showing that changing the way you think can profoundly improve your psychological health and well-being. Psychological health refers to a person’s social, emotional, and mental well-being. It encompasses many facets including emotional regulation, communication, decision-making, relationship satisfaction, as well as the ability to cope with stress and work productively. Psychologically healthy individuals typically manage their emotions effectively, have a positive sense of self, make sound decisions, and maintain healthy relationships with others. Decades of research have shown that errors in the way we think, called cognitive biases, have a profound impact on our psychological health. Fortunately, the ability to minimize many cognitive biases has been well-documented. Yet, individual research projects tend to focus on one specific bias, one specific debiasing approach, or one aspect of psychological health, with limited communication and integration across disciplines. For example, research on how cognitive biases can be reduced to improve decision-making has primarily been the purview of cognitive and social psychologists or behavioral economists (e.g., [5-8]); whereas, essentialism bias and mindset modification have roots in developmental psychology (e.g., [9,10]) and research on modifying negativity bias is most often studied by clinical psychologists treating depression and anxiety (e.g. [11-15]). In a similar vein, most manuscripts also focus on discussing one type of bias, and its implications for one area of research.

In this manuscript we bring together research from various disciplines to provide a more complete account of how changing the way we think can improve psychological health. First, we review evidence on how different cognitive biases—negativity bias, framing bias, confirmation bias, and essentialism bias—can impact several aspects of psychological health. Next, we review the cognitive biases that interfere with social perspective taking to impact well-being. Throughout our review we highlight the tremendous benefits of mitigating cognitive biases for several facets of psychological health. Our overarching aim is to make the case for integrating multiple evidence-based strategies from different disciplines because we believe that combining these strategies is the best way to maximize social-emotional health.

Minimizing Cognitive Biases to Maximize Social-Emotional Health

Cognitive biases are normal by-products of how the mind works, yet vast individual differences in the magnitude of these biases predict myriad aspects of psychological health and quality of life including, but not limited to, decision-making abilities, interpersonal skills, relationship satisfaction, stress management, workplace productivity, academic achievement, self-esteem, and mental illness [10,16- 18]. One prominent example of the link between cognitive biases and psychological health comes from clinical research showing evidence that depression, stress, and anxiety are all associated with negativity bias. Negativity bias is the tendency for negative events, information, or emotions to weigh more heavily in our minds than positive ones. For example, if you have ten good experiences and one bad one, you’re likely to remember the bad one more vividly and let it affect you more deeply (e.g., [19,20]). The lion’s share of research in this area has been conducted by clinical psychologists focusing on two categories of negativity bias; attentional bias and interpretation bias. Attentional bias is characterized by the preferential allocation of cognitive resources (e.g., attention) to negative stimuli. Numerous studies have demonstrated that individuals with depression show a heightened attentional bias toward negative information. For instance, research has shown that depressed individuals are faster to detect and respond to negative stimuli compared to neutral or positive stimuli [15]. This bias is thought to perpetuate and intensify negative mood states by reinforcing the focus on distressing information.

Interpretation bias involves the tendency to interpret ambiguous or neutral information in a negative manner. This type of bias has also been linked to depression. Experimental evidence reveals that individuals with depression are more likely to interpret ambiguous situations negatively compared to non-depressed individuals (e.g., [21]). This bias is also associated with higher levels of stress and anxiety. Research indicates that negativity bias exacerbates anxiety symptoms by skewing the perception of everyday stressors as more threatening than they really are [22]. For instance, a study by MacLeod and Mathews [23] found that individuals with a strong negativity bias are more likely to experience heightened anxiety and stress, as they tend to focus on potential dangers and threats. Importantly, research suggests these biases are not merely associated with mood disorders but actively contribute to their development and maintenance (e.g., [13,14,24]). For example, one study found that individuals with a negative attentional bias were more likely to experience longer- lasting depressive symptoms over time [13]. These findings are further supported by prospective studies indicating that even among nonclinical samples, such as university undergraduate students, greater biases predict elevated depressive symptoms months later [25-28]. Critically, training aimed at reducing these biases has been shown to lessen the severity of depressive symptoms from pre- to post-training in individuals with a history of depression [29,30].

Notably, negativity bias does not operate in isolation to affect psychological health. Research shows that other cognitive biases can reinforce or amplify negativity bias and exacerbate pessimistic thinking, stress, and dissatisfaction [31]. Although dozens of cognitive biases can impact psychological health (e.g., [32-34]), we highlight three more in this section with clear health consequences, namely confirmation bias, framing bias, and essentialism bias. Confirmation bias is the tendency to search for, interpret, and remember information that confirms one’s preconceptions [35]. Confirmation bias can work in tandem with negativity bias to exacerbate pessimistic thinking, stress, and dissatisfaction and reinforce negative beliefs. As an example, a study found that individuals who hold negative biases about their partners are more likely to interpret their partners’ actions in a negative light, which can erode relationship satisfaction [31]. Similarly, another study found that children with higher levels of worry tended to seek information confirming danger and avoid information disconfirming it, showing how confirmation bias can amplify anxiety by promoting a skewed perception of threats [36].

As a different example of how confirmation bias can impact health, consider how it impacts the diagnostic accuracy of healthcare providers. Research showed that approximately one in eight physicians and one in four medical students exhibited confirmation bias when gathering new information after forming initial diagnoses [37]. This tendency to seek confirmatory evidence not only increased the likelihood of incorrect diagnoses, but also impacted future therapeutic decisions. This example reiterates the point that everyone can benefit from learning strategies to minimize cognitive biases. Fortunately, researchers have identified promising interventions to mitigate the effects of many cognitive biases, including confirmation bias. For instance, Morewedge et al. [38] found that a single session of training, involving education about cognitive biases via videos or interactive computer games, significantly reduced confirmation bias (among other biases). These reductions were observed immediately and were still present at least two months later (see also [39,40]).

Another cognitive bias with psychological health implications is framing bias. Framing bias refers to the cognitive tendency to respond differently to the same information depending on how it is presented or “framed”. This bias significantly impacts decision-making, risk perception, health behaviours, and consumer choices. Framing bias has been extensively studied in the context of financial decision-making and marketing (e.g., [41,42]). In health contexts, framing bias affects how individuals perceive risks and make health-related decisions. For instance, studies have shown that people are more likely to accept treatment options when the benefits are framed positively (e.g., “90% survival rate”) rather than negatively (e.g., “10% mortality rate”) [43]. Similarly, framing bias also impacts adherence to medical treatments and health regimens. Research indicates that patients are more likely to comply with medical advice when the benefits of adherence are framed positively. For example, presenting a medication’s benefits in terms of enhanced quality of life, rather than in terms of avoiding negative outcomes, has been shown to increase adherence rates [44]. Framing bias also influences decisions related to vaccinations, health screenings, dietary choices, and lifestyle options, as just some examples (e.g., [45,46]. Clearly, the way information is communicated can either hinder or enhance efforts to improve public health.

The effects of framing bias are not limited to decision-making—it also profoundly affects our emotional responses. Individuals are more likely to experience positive emotions when information is framed positively, and negative emotions when it is framed negatively. This dynamic has significant implications for therapeutic practices as well as preventative approaches. Cognitive Behavioral Therapy (CBT), for example, employs cognitive reframing techniques to help individuals identify and challenge negative thought patterns, replacing them with more constructive perspectives. CBT has been shown to be highly effective in clinical settings (see [Hofmann et al (2012) for a meta- analysis). Similarly, other research indicates that reframing stressful events in a more positive or controllable light can reduce perceived stress and enhance coping mechanisms (e.g., [47]). The concept of learned optimism [48] also capitalizes on reframing techniques. Learned optimism approaches teach individuals to adopt a more optimistic explanatory style by reframing adversities as opportunities for growth and challenging and reframing pessimistic beliefs. These approaches reduce depression and anxiety while improving emotional regulation and overall well-being (e.g., [49]), underscoring the importance of addressing framing bias to enhance resilience.

Essentialism bias is yet another cognitive bias that impacts well-being in several ways. Essentialism bias refers to the cognitive tendency to view certain categories or groups as having an underlying, unchanging essence that defines their characteristics [9]. This bias can lead individuals to believe that one’s attributes are inherent and immutable rather than subject to change [9] and can lead to stereotyping and prejudice towards social groups [50,51]. People also show essentialism bias regarding their own characteristics. In this sense, this bias is closely linked to the concept of a fixed mindset— the belief that human traits, such as intelligence, are innate and unchangeable [10]. A fixed mindset can hinder personal development and resilience because individuals who hold this mindset perceive their traits and abilities as largely unchangeable, reducing their motivation to seek improvement. A growth mindset it the opposite of a fixed mindset and is best described as the belief that one’s traits, attributes, or abilities can be shaped through effort [10].

Our mindset permeates nearly every facet of our personal and interpersonal experiences (e.g. [10,52]). For example, studies have shown that possessing a growth mindset enables psychological resilience in the face of negative life events [53]; see also [54,55]. This mindset is also positively correlated with perceived control and self-efficacy in health behavior [56]. In contrast, people with a fixed mindset are more likely to experience anxiety and depression in response to failure [57]. The key to this association may be in the improved coping strategies that individuals with growth mindsets demonstrate [52,58]. Research has shown that individuals who hold a growth mindset are more willing to learn and more likely to seek and adopt coping strategies than those who hold a fixed mindset [52]. In other words, individuals who hold a growth mindset believe that they can adapt to a difficult situation, whereas someone who views their abilities as fixed tends to avoid challenges. Furthermore, individuals who hold a fixed mindset report greater shame and stress in the face of failure and are more likely to blame themselves [52-59]. In contrast, individuals with a growth mindset are shown to be more optimistic, believing that people can change and improve [10]. A 2024 study with middle-school adolescents revealed that higher growth mindset predicted greater psychological resilience and mediated the relationship between mindset and mental health [60].

Fortunately, a wealth of research has shown that people can be readily taught to be more ‘growth-minded’. Recent research on growth mindset interventions highlights the effectiveness of even brief online programs. One such intervention, ‘Learning Mindsets’ (delivered in 2 online sessions in under an hour) showed significant positive effects on students’ mindsets [59]. Similar short interventions were also effective at promoting a growth mindset, even more than some longer in-person approaches, suggesting that concise and accessible formats may be particularly impactful [61]. Importantly, neurocognitive research reveals that adopting a growth mindset can mitigate the negative effects of depression on cognitive abilities [62] and encourage individuals to accept, and utilize, critical feedback [63]. The willingness to adopt positive coping strategies such as acceptance, as opposed to rumination or self-blame, has a strong impact on the social and emotional well-being of individuals facing negative life events. For example, a study examining cancer patients and individual coping strategies found that those practicing adaptive strategies such as modifying uncomfortable situations showed greater resilience and psychological adjustment [64]. Another study showed that breast- cancer patients who used adaptive coping styles reported fewer depressive symptoms [65]. Consistently, cultivating a growth mindset and teaching adolescents to adopt positive coping strategies has also been shown to improve adolescents’ mental health [58]. Notably, addressing these biases early in development has the greatest potential to prevent social-emotional problems and yield the most long-term benefits.

Improving Perspective Taking to Maximize Social- Emotional Competence

Social perspective taking, the ability to infer and reason about others’ mental states (e.g., their knowledge, beliefs, intentions, desires, thoughts, and emotions; sometimes called ‘theory of mind’ or mentalizing) is a core component of psychological competence. Perspective taking enables individuals to appreciate diverse viewpoints, empathize with others, and understand the impact of their actions on others (e.g., [66,67]). Research has consistently shown that perspective taking abilities are involved in virtually every social interaction and are critical for effective communication, social decision-making, and maintaining social relationships (e.g., [68-70]). For example, perspective taking is associated with higher levels of empathy, prosocial behavior, and social understanding, which can lead to reduced interpersonal conflicts and increased relationship satisfaction [71]. For instance, studies by Peterson et al. [72] found that perspective taking is associated with increased self-esteem and higher quality friendships. More advanced social perspective taking also appears to act as a protective factor against trauma and adversity (e.g., [73-75]). Conversely, poor perspective taking skills are associated with greater psychological distress [76], more emotional symptoms, and increased loneliness [77]. This latter result is especially noteworthy given longitudinal studies linking loneliness to a variety of negative health outcomes, including poorer sleep quality [78], and increased depressive symptoms [79]. For instance, a meta-analysis of 18 studies examining the relationship between social perspective taking and Major Depressive Disorder in adults revealed that deficits in perspective taking can be a risk factor for depression and psychosocial impairment, with the level of perspective taking problems predicting symptom severity [80].

Not surprisingly, the way we think about others and their mental states (i.e., perspective taking) is also vulnerable to cognitive biases. Of particular interest in this manuscript is the category of cognitive biases called perspective-taking biases (sometimes called ‘egocentric biases or social cognitive biases). Perspective-taking biases, systematic tendencies or errors in the way we think about others’ mental states (or perspectives), can be particularly damaging to interpersonal relationships, impair communication, and lead to poor social decision-making (e.g., [6,81,82]). One perspective taking bias, the curse of knowledge bias, refers to the tendency to be swayed by one’s knowledge when reasoning about a more naive perspective (e.g., [7- 8,83-87]). A classic example of the curse of knowledge bias (sometimes called ‘hindsight bias’) is when adults who know the outcome of an event (e.g., a sports game, an election, or a battle) overestimate how likely others are to predict that outcome. In contrast, adults who are unaware of the event’s outcome tend to make more accurate estimates of what others will predict (e.g., [88-91]). This bias has been shown to affect judgements and decision making across a wide range of contexts including medicine, education, politics, law, business, and economics (e.g., [6,92-94]; see [91,94] for reviews). In education, for example, teachers with knowledge of the subject matter they are teaching often overestimate how clear their lessons are for students (e.g., [92]). This bias affects communication and social judgments in various ways because it causes individuals to overestimate the likelihood that others share their knowledge. Given the regularity with which we must gauge what others know, this bias frequently leads to miscommunication and misunderstandings in everyday conversations as well as formal communications (e.g., [68,83,95-96]). These communication breakdowns can create conflict and stress and may impact an individual’s self-esteem if repeated miscommunications make them question their ability to relate to others. A related perspective taking bias, the false consensus effect, is the tendency for people to overestimate the extent to which others share their beliefs, opinions, and behaviors [97]. In other words, individuals often assume that most people think or behave the same way they do, negatively influencing decision-making, social interactions, and group dynamics. For example, when individuals assume that others share their views, it can lead to disagreements and conflict when they realize their opinions differ. This effect can similarly lead to distorted perceptions of social norms and contribute to problems in group decision-making and impair group cohesion [97-101].

Another perspective taking bias, the spotlight effect, occurs when individuals overestimate the extent to which others notice and evaluate their actions and appearance. This can lead to heightened self- consciousness and increased social anxiety, as individuals mistakenly gauge the level of social scrutiny they will receive. Gilovich et al. [102] demonstrated this in a study where participants consistently overestimated the attention they received—believing that twice as many people would remember the embarrassing T-shirt they wore, compared to the actual number who remembered. In a follow-up study, participants also overestimated the likelihood their classmates would notice even minor fluctuations in their physical appearance [103]. This effect occurs across many different contexts from volleyball games to video games. Consistently, participants overestimate how much their teammates will notice their performance flaws and expect more critical feedback than they receive [102,103]. This tendency for individuals to feel that they are the center of attention is linked to increased self-consciousness and social anxiety, directly affecting their psychological health. For instance, researchers found that socially anxious individuals were more likely to exhibit the spotlight effect, reporting heightened anxiety and evaluating their performance more harshly when they felt observed by others [32].

Another bias related to perspective taking is the fundamental attribution error (FAE). FAE is a cognitive bias that leads individuals to overestimate how much another person’s behavior or circumstance is due to their personal character (i.e., their ‘fundamental nature’), rather than considering the influence of external (situational) factors on their actions [104]. In a classic example of FAE, participants listening to a speech believed that the speaker’s personal beliefs aligned with their presentation even when they were explicitly told the speaker’s position was “decided by a coin toss” [105]. Participants disregarded the situational constraints and tended to assume that the speech was based on personal beliefs and traits [105]. Some researchers suggest that this tendency arises because people find it simpler to attribute a person’s actions to their personal characteristics [106,107]. For example, researchers have argued that in the context of people’s misfortunes, it is easier to blame an individual for their circumstances, by attributing their misfortunes to their personal characteristics, actions, and choices rather than considering more complex contextual factors [106,107]. This process appears to emphasize personal responsibility and foster victim blaming [108]. Consequently, FAE can contribute to increased judgments of others and reduced compassion [97].

Perhaps not surprisingly, perspective taking offers a powerful countermeasure to the FAE. Perspective taking encourages people to adopt the viewpoint of the other person to consider the situational factors contributing to their actions before blaming the individual. That is, by reasoning about another person’s mental states, individuals can better understand their point of view and acknowledge the situational constraints that affect their actions and decisions. Thus, perspective taking may lead to a reduced sense that the other person is accountable, especially towards victims of circumstance, such as those trapped in a systemic cycle of poverty. For example, Hooper et al. [104] demonstrated that a brief perspective-taking exercise focusing on shifting perspectives significantly reduced the FAE. Participants who completed the exercise were better able to attribute behaviors to situational factors rather than dispositional traits (see also [109]). In other words, perspective taking seems to shift the blame from individuals to broader situational factors, fostering a more empathetic view of behavior. Following a similar logic, researchers should be able to reduce the FAE, and other perspective taking biases, through a range of activities that enhance perspective- taking (e.g., [110-112]).

To date there are several promising methods for enhancing perspective-taking. Research in this area has taken one of two general approaches. One general approach involves highlighting the different kinds of thoughts, emotions, and opinions people have in different contexts, depending on their unique experiences, backgrounds, and predispositions. There are several different types of techniques used in this area of research; collectively we call this type of approach the ‘Alternate Views’ approach. This general approach can be conducted passively (e.g., by exposing individuals to scenarios, real or hypothetical, with a range of different viewpoints) or actively (e.g., asking individuals to engage in real or imagined role-taking exercises, or reflect on their own thoughts and actions in different contexts and how those actions might be perceived by others (e.g., [113-115]). For example, Rezaei et al. [109] found that medical students who engaged in these reflective practices (e.g., via journaling), improved their empathy and ability to adopt patients’ perspectives. Other work has examined the efficacy of using acting lessons to foster perspective taking [116]. A wealth of other research has highlighted how increasing mental state discourse (i.e., simply talking more about mental states and differing points of view) can improve perspective taking abilities (for a review see [117]).

A second general approach that has been used to improve perspective taking is the Cognitive Debiasing approach which directly targets the biases that can impede perspective-taking. This approach involves educating individuals about common cognitive biases and strategies for minimizing them (e.g., [82]). Notably, cognitive debiasing methods are not specific to enhancing perspective taking but are commonly used to reduce cognitive biases to improve decision- making across a range of contexts (e.g., [38-39,102,118,119]). Given the many benefits of enhancing perspective-taking, we believe that approaches for enhancing psychological health should incorporate strategies for improving perspective taking as often as possible. In our opinion, the most effective interventions to foster psychological health will capitalize on the benefits of the Alternate Views approach and the Cognitive Debiasing approach. That is, we believe optimal results can be achieved by integrating the alternate views approach with education about cognitive biases and strategies for minimizing them.

Closing Remarks

Effectively addressing the mental health crisis will require a comprehensive, multi-pronged, approach. While piecemeal approaches provide valuable insights into the individual factors that influence well-being, overcoming the mental health crisis will require multiple evidence-based strategies. Individuals experience psychological health problems for a myriad of reasons (e.g., [120]). The kind of treatment or strategy that works for one person may not work for everyone. Combining strategies is the best way to address the unique and diverse challenges individuals face to maximize the benefits for individuals and society.

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Pheromonicin: A Novel Antimicrobial Inhibited Fatal Multidrug-Resistant M. tuberculosis Infection in Animal Models

2 Replies

DOI: 10.31038/MIP.2024521

Abstract

There is an urgent need for effective new drugs to combat multidrug-resistant M. tuberculosis (MDR-TB). We found the outer membrane porin A of MDR-TB (OmpATb), a member of the OmpA family which shares epitopes with a variety of microbes, could be targeted by a 28-residue antibody mimetic by fusing two antibody Fab complementarity-determining regions (VHCDR1 and VLCDR3) through a cognate framework region (VHFR2) of a monoclonal antibody which recognizes OmpA of N. meningitidis. We constructed a fusion protein, pheromonicin-NM (PMC-NM), by linking colicin Ia, a bactericidal molecule produced by E. coli which kills target cells by forming a voltage-dependent channel in target cell membrane, to that antibody mimetic. The OmpATb/antibody mimetic interaction initiated the formation of irreversible PMC-NM channel in MDR-TB cell membrane resulting in leakage of cellular contents. PMC-NM demonstrates high efficacy, 103-5 times greater than that of current anti-TB agents, against 506 isolates of MDR-It can reduce pulmonary TB CFU 2-3 logs compared to controls in murine TB models. With a 22-wk PMC-NM treatment, 75% macaques survived MDR-TB infection that was lethal to untreated and INH/RIF-treated controls. No relapse occurred in the subsequent 26-wk observation period for 60% of the macaques. PMC-NM significantly altered outcomes of in vivo fatal MDR-TB infection without evident toxicity, making it an appropriate candidate for further clinical evaluation.

Keywords

OmpATb, Guidance of antibody mimetic, Cellular leakage, Intracellular clearance, Pulmonary TB burdens, Clinical outcomes

Introduction

Tuberculosis (TB) is the biggest infectious killer of adults, causing approximately 1.8 million deaths and 10.4 million new cases each year [1-4]. Multidrug-resistant tuberculosis (MDR-TB), caused by strains of Mycobacterium tuberculosis resistant to at least isoniazid (INH) and rifampicin (RIF), the backbone of the current empirical first-line anti-tuberculosis regimen, affects an estimated 600,000 people every year. Cases of extensively drug- resistant tuberculosis (XDR-TB), resistant to INH/RIF, fluoroquinolone, and amikacin, capreomycin or kanamycin, have been observed worldwide, most notably in high burden countries such as China, India, throughout Africa, and Eastern Europe [1-4]. Current therapies for drug-resistant tuberculosis offer a very low cure rate – roughly 50 percent – and therapy ranges from 9 months to two years, therefore, new drugs are urgently needed to combat MDR/XDR-TB infections [4].

Outer membrane porin A of M. tuberculosis (OmpATb), located on the outer membrane of Mtb cells [5-8], could serve as a potential target for new drugs. We postulated that OmpATb might appear on the surface of infected host cells [9,10] If that was the case, the new drug would be able to kill both Mtb cells and infected host cells.

As an example of channel-forming bacteriocins, colicin Ia, a typical E1 family colicin, is bactericidal to E. coli and might be able to be developed as a novel candidate against MDR-TB infection, if the native targeting ability of wild-type colicin Ia could be altered. Colicin Ia kills target cells by forming a voltage-activated channel in the target cell membrane via its 175-residue C-terminal channel- forming domain [11-16]. It acts on the lipid bilayer of cell membranes, therefore, colicin Ia could be engineered for insertion into the cell membrane of bacteria that are not its natural targets [18]. In target cell membrane, the active form of colicin Ia is a monomer [12-16].

To target the channel-forming domain of colicin Ia to the cell membrane of other bacteria, we initially constructed fusion proteins consisting of either an 8-residue staphylococcal AgrD1 pheromone, or a 7-residue enterococcal cCF-10 pheromone, or a 13-residue Candida a-factor pheromone fused to the channel-forming domain of colicin Ia. The fusion proteins demonstrated effective bactericidal activities against methicillin- resistant Staphylococcus aureus, or vancomycin- resistant Enterococcus faecalis, or Candida albicans in vitro and in vivo, respectively [17-19]. We then selected certain antibody complementarity- determining regions (CDRs) and framework region sequences to create a single-chain antibody mimetic comprising two interacting VH– and VL– derived CDRs. The mimetic retains the basic antigen-recognition ability of the whole parent antibody and acts as a smaller, proper-affinity binder [20]. We previously found that the most promising structure comprises VHCDR1 and VLCDR3 connected by a corresponding VHFR2 sequence, forming a 28-residue antibody mimetic [20].

Materials and Methods

Construction and Purification of Pheromonicin-NM

The antibody mimetic amino acid sequence was constructed to followpositionI626 ofcolicin Iabydouble-strandedmutagenesis(Quick Change kit, Strategene) using a pET-11 plasmid containing the colicin Ia gene to form pheromonicin-NM (PMC-NM). The oligonucleotide used, containing the desired SYWLHWIKQRPGQGLEWIGSQS

THVPRT mutation, was 5’-GCG AAT AAG TTC TGG GGT ATT TCT TAT TGG CTG CAT TGG ATT AAA CAG TAA ATA AAA TAT AAG ACA GGC-3’, 5’-TGG CTG CAT TGG ATT AAA CAG AGA CCT GGT CAG GGA CTG GAA TGG ATA TAA ATA AAA TAT AAG ACA GGC-3’ and 5’- GGT CAG GGA CTG GAA TGG ATA GGA TCT CAG TCC ACG CAT GTG CCG AGA ACC TAA ATA AAA TAT AAG ACA GGC-3’. The harvested plasmid was transfected into pET BL-21 (DE3) E. coli cells to produce PMC-NM as previously described10-13. As determined from 12% SDS-polyacrylamide gel assays, PMC-NM eluted by 0.3 M NaCl comprised about 90% of total eluted protein (Figure 1d).

Amino acid Sequences of Mimetics of Other Tested Pheromonicins

The sequences of the VHCDR1-VHFR2-VLCDR3 of the following parent molecules are:

HB8627 IgM recognized non-small cell lung cancer: DYYLHWVKQRTEQGLEWIGQHIRELTRS

IgG recognized B. dendrobatidis: GYTMEWVKQSHGKN- LEWIGQNGNTLPYT

AF468835 recombinant single chain Fv Ab recognized N. gonor- rhoeae:

TYAMNWVRQAPGKGLEWVAQQGQSYPLT

IgG recognized Cyanobacteria: SYWMQWVKQRPGQGLEWIGQ- QYWSTPPWT

MDR-TB Isolates Used for In vitro and In vivo Rodent and Primate Infection

506 clinical isolates of MDR-TB were collected by China CDC from southern and western China with 10% XDR-TB isolates for in vitro study. Mtb Erdman, H37Rv, MDR-06005 and PUMC-94789 were used for rodent and primate infection models in the present study. MDR-06005 is a Beijing genotype strain that is katG 315 and gyrA 281 genetic mutations (isolated by Institute of Tuberculosis Research, 309 Hospital, Beijing). PUMC-94789 is a Beijing genotype strain that is with katG 315, rpoB 531, 526 and gyrA 94 genetic mutations, and presented a 7-d median death time in mice test (isolated by Institute of Experimental Animal Sciences, Peking Union Medical College).

Minimum Inhibition Concentration

100 μl of double-diluted preparations of either PMC-NM, INH, or other routine anti-TB agents was added into respective wells of 96- well plate while only 100 μl 7H9-S broth was added into the control well, then 100 μl of inoculum (TB cells, 106CFU/ml in 7H9-S broth) was added into each well. The plates were sealed in plastic bags and incubated 10 days (37°C) after inoculation. The lowest concentration of each agent which prevented the visual growth of Mtb cells was interpreted as MIC. MIC measurement of PMC-NM against isolates of Gram-positive and -negative Omp A bacterium were performed at Antibiotic Division, National Inst. for Food and Drug Control. China FDA with a paid service contract. Incubation time was 12-24 hrs (37°C) after inoculation.

In vitro Inhibition Test

100 μl of preparations of PMC-NM, or INH, or wild-type colicin Ia, or Rev-PMC-NM was added into respective wells of 96-well plate while only 100 μl 7H9-S broth was added into the control well, then 100 μl of inoculum (H37Rv cells, 106CFU/ml in 7H9-S broth) was added into each well. The plates were sealed in plastic bags and incubated 2, 3, 7, 8 days (37°C) after inoculation. Turbidometric absorbance of each well was measured.

Phase I incubation test, 3 ml 7H9 solution inoculated with H37Rv (0.1 ml, 104-5 CFU), treated with (1) 50 mM borate buffer (colicin Ia and PMC-NM stock solution), (2) 1 ug/ml wild-type colicin Ia, (3) 1 ug/ml PMC-NM, incubated at 37°C for 6 weeks.

Phase II incubation test, 3 ml 7H9 solution inoculated with H37Rv, treated with (1) nothing, (2) 50 mM borate buffer, (3) 2 ug/ml wild-type colicin Ia, (4) no inoculation but remains of phase I PMC- NM treatment culture (3,000 rpm centrifuged 10 min), incubated at 37°C for 10 weeks.

Bifidobacterium longum (1.2186) and Lactococcus lactis (1.2472) (Inst. Microbiology, China Academy of Science) (50 μl, 103-4 CFU/ml) was smeared on the surface of respective solid medium with PMC- NM and ampicillin treatments, incubated 18 hrs (37°C).

Rodent In vivo Bactericidal Activity

Female BALB/C mice (six per group) were infected intranasally with 6.8 x 102 CFU of Mtb Erdman (ATCC35801). Twenty-one days post-infection mice were treated with either intraperitoneal injection (i.p.) of saline, or PMC-NM 20 or 40 mg/kg/d, or oral delivery of INH 25 mg/kg/d for 4 weeks. Right lungs were collected for Mtb CFU determination.

Female BALB/C mice were injected with 0.2 ml 105-6 CFU/ ml of MDR06005 MDR-TB strain through the tail vein. Three days after infection, mice were randomly assigned into four treatment groups (twenty per group) and i.p. with 0.9% saline alone, INH 6, or 12, or 27 mg/kg/d, PMC-NM 5, 10, or 20 mg/kg/d daily for 4-weeks. The number of surviving animals at various time points was determined. Kaplan-Meier analysis was used to determine the significance of differences between the PMC-NM and control groups. All in vivo protocols were approved by the Institutional Animal Care and Use Committee of Sichuan University and Project of Sichuan Animal Experimental Committee, License 045, Institutional Animal Care and Use Committee of China CDC and Project of Beijing Animal Experiment Committee, license CNAS BL0047, China, and Subcommittee for Animal Safety, VAMC, Syracuse, NY.

Immunolabeling Assay

Pheromonicin-NM was injected i.p. into mice, or macaques that had been infected with MDR-TB cells 4-6 weeks (mice), or 17 weeks (macaque) prior. The animals were killed 12-24 hrs after injection, and the lungs were fixed for formalin and paraffin-embedded prior to sectioning. Sections were sealed with 5% BSA, and then incubated with anti-OmpA (rabbit IgG, MyBioSource, San Diego, USA), or anti- pheromonicin (mouse, AbMax, Beijing) antibodies for 30 min. at 37°C, washed and incubated with FITC- anti-rabbit and rhodamine- anti-mouse goat antibodies (Bioss, Beijing) for 15 min. at 37°C, and washed. The sections were examined under an optical/fluorescent microscope (Nikon 90i) with DM400, DM505 and DM565 filters.

MDR-TB infected macaque lung tissues were fixed with 2.5% paraformaldehyde, dehydrated with gradient concentrations of ethyl alcohol, embedded in gelatin, and sectioned with Leica EM UC6 ultramicrotome, ultra-thin sections were probed with above rabbit anti-OmpA Ab and mouse anti-pheromonicin Ab as first Ab, 2nd Ab were goat anti-rabbit 5 nm colloidal gold and goat anti-mouse 10 nm colloidal gold IgGs, (Sigma), stained with uranyl acetate and observed under a transmission electron microscope (Hitachi HT7700, Japan).

Immunoblotting Assay

Pellets of H37Rv Mtb strain (2 gm) and macaque lung tissues (2 gm, extracted from autopsy as CFU counting collection) were homogenized, suspended with extract solution A (9 ml with 1: 250 extract sol. C), sonicated for 1hr at 2-8°C, centrifuged 10,000g, 5 min at 2-8°C, supernate suspended with 1: 30 extract solution B, 37°C bathing for 10 min, centrifuged 1,000g, 3 min at 37°C, the thick liquid (bottom layer of layered centrifugation) blended with 1: 1 extract sol. B (membrane protein extraction kit, BestBio, Shanghai). 60 ug preps per well, SDS-10% PAGE electrophoretic transferred to nitrocellulose membrane, incubated with, or without 10 ug/ml PMC-NM, or other tested pheromonicins for 1 hr, 20°C, then 4°C over night, the resulting material were probed with anti-pheromonicin Ab and appropriate secondary antibodies (AbMax, Beijing).

Ethics Statement

The work described in this study was carried out at Center for Primate Biomedical Research, University of Illinois College of Medicine, Chicago and Institute of Experimental Sciences, Peking Union Medical College via paid service contracts, and the Veterans Affairs medical center, Syracuse, NY). All animals were housed in the Animal Bio-Safety Level III (ABSL-III) facility located at Biologic Resources Laboratory, University of Illinois at Chicago and Institute of Laboratory Animal Science, Peking Union Medical College. The housing and animal care procedures were in compliance with the Chinese guidelines for animal experiments (Laboratory animal Requirements of environment and housing facilities GB 14925–2010, China; Regulations on administration of laboratory animals, Ministry of Science and Technology, 1988, China) and with the 8th Guide for the Care and Use of Laboratory Animals of Association (National Research Council, 2011). The animal housing and care procedures were in compliance with the United States Department of Agriculture (USDA) through the Animal and Plant Health Inspection Service under the Animal Welfare Act (AWA). The ABSL-III facility was certified by USDA and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) prior to initiation of this project. Office of Animal Care and Institutional Biosafety (OACIB) of University of Illinois at Chicago approved all the study protocols and procedures before this project begins. The approval numbers were: IBC#12-089; ACC#12-210. The ABSL-III Laboratory is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Institutional Animal Care and Use Committee of Peking Union Medical College approved study protocols and procedures prior to starting this project (ILAS-PC-2014-009). Animal protocols were approved by the Subcommittee for Animal Studies (SAS), Veterans Affairs Medical Center, Syracuse, NY. Animals were individually housed in stainless steel wire-bottomed cages (80×80×80 cm3) with sufficient space supplied with a commercial monkey diet and water and twice daily with fresh fruit in an air-conditioned room and monitored by a computer-based system. After infection, animals were also monitored twice daily by experienced staff. Animal health was monitored daily by the animal care and veterinary personnel. Pre-defined humane endpoints including depressed or withdrawn behavior, abnormal respiration rates, serious loss of appetite, severe body weight loss and severe abnormal radiographic changes were applied to reduce discomfort in this study. Serious loss of appetite was defined as no food intake during at least two meals. Severe body weight loss was defined as a 20% weight loss in three consecutive weeks compared to the body weight before TB infection. Animals were euthanized by anesthesia with ketamine (i.m. 3～5 mg/kg).

Study Plans

Cynomolgus macaques (Macacca fasicularis) were infected with MDR-TB PUMC-94789 via bronchoscope and monitored clinically, as previous described. Upon development of active TB, animals were randomly assigned to one of three groups: untreated, combind INH/ RIF, or PMN-NM. Animals were followed for 22-wk treatment and subsequent 26-wk observation period. Gross pathology, overall bacterial burden scores, and lung CFU were calculated as described for treatment response. CT scans were performed (a) before infection as the baseline background, (b) day 22 after infection to confirm infection, (c) at time points of 22-wk treatment and subsequent 26-wk observation period and (d) at the end of treatment or termination of animal. Cynomolgus macaques were infected with M. tuberculosis H37Rv via bronchoscope and underwent clinical observation, necropsy and histology.

Clinical Observation, Necropsy and Histopathology

Clinical assessments included body weight, respiratory distress, general alertness/activities and food uptake. Clinical lab tests included CBC, differentials, calculation of total lymphocyte population and serum biochemical examination. Extensive quantification of gross TB lesions in each lung lobe, hilar lymph nodes, pleural and extra thoracic organs was undertaken. Histopathology of TB lesions from right caudal lobe (infection site) and other lobes, chest lymph nodes, and extra thoracic organ/tissues was evaluated.

Tissue Bacterial Burden Measurement

Lung samples (4-5 gm) were collected at necropsy from upper, middle and caudal lobes of left and right lungs. Samples were homogenized with a grinder, and digested in N-acetyl-L-cysteine- NaOH-Na citrate (1.5% final concentration) for 15 min at room temperature, neutralized with phosphate-buffered saline (PBS, 67 mM, pH=7.4) and centrifuged at 4,000 g for 15 min. Sediments were re-suspended with 2 ml PBS buffer. 100 μl of each 10-fold serial dilution was inoculated on 7H11 agar plate, and then incubated at 37°C for 4 weeks. The bacteria burden was calculated according to the number of CFUs on the agar plate.

CFU of organ (lung) was calculated as, [CFU number of agar plate x 10 (10-fold serial dilution)/[weight of sample (mg)/1,000 (gm) ]] x weight of lung (gm) CFU/lung

CT Scanning and Abnormal Volume Measurement

Image acquisition. All scans were acquired with a 16-section CT scanner (μCT S-160 scanner, United Imaging, Shanghai) by using a spiral mode with 16 × 0.9375-mm collimation. Exposure settings were 140 kVp and 200 mA. Transverse images were reconstructed at a thickness of 1.0 mm and a 1.0-mm increment by using a moderately soft reconstruction kernel, the smallest field of view that included the outer rib margins at the widest dimension of the thorax, and a 1024 × 1024 matrix. All images were interpreted by radiologists who were blind to the identity of the subject. Semi automated measurements of Pulmonary Abnormal Volume (PAV). Data were transferred from the CT scanner to a digital workstation (United Imaging, Shanghai) with commercially available software for semi-automated PAV measurements (Lung lesions; Tissue Management). Lesions were identified by using transverse thin-slab Maximum Intensity Projections (MIP) that were displayed with window width and level settings of 1500 and −500 HU, respectively. With the candidate lesion was marked, the program automatically looked for adjacent structures which matched conditions around the lesion, started volume measurement by using volume-rendered with the structure and the volume of segmented lesion was calculated. The CT scan which taken before TB infection was used as the baseline.

Statistical Analysis

Comparison of data among experimental groups were performed by t test. Comparison of cumulative survival among experimental groups were performed by Kaplan-Meier analysis. P values of <0.05 were considered to be statistically significant. The following inclusion criteria were used to select macaques: no infectious or chronic diseases, good appetite and behavior, age 2-4 yrs, bodyweight 4-6 kg. The macaques were individually housed in cages for five weeks before pulmonary MDR-TB infection. Three weeks after infection with confirmation of eyelid tuberculin test and pulmonary CT scan, animals were randomly allocated into three treatment groups according to a Blocked Randomization Method.

The sample size was estimated according to following assumption: Group I (placebo control) and Group II (traditional medications control) would have an effect of survivor rate equal to 0%, respectively. Group III (PMC-NM) would be a 50% of survivor rate after a 22- wk treatment intervention (25-wk pulmonary infection). When we combined Group I and Group II into a control group to conduct difference comparing of two survivor rates (0% and 50%) between control group (8 animals) and experiment group (8 animals), and defining alpha=0.05, beta=0.2, a Fisher exact test P value=0.038 under selecting n1=8 and n2=8.

Data from our study showed that the survivor rate of PMN- NM treated group was 75% (6/8,) and the survivor rate of control and traditional medications group was 0% (0/4, 0/4), a Fisher exact test P value=0.030 (one-side), it indicated significant statistical difference. The numbers of surviving animals at various time points were determined, and Kaplan-Meier analysis showed a significant difference between the control and PMC-NM treated group (p=0.003). Therefore, we assume that the sample sizes of our study are sufficient.

Results

PMC-NM demonstrated high efficacy against pan-susceptible and MDR-TB strains under the guidance of 28-residue antibody mimetic.

We fused the antibody mimetic at the C-terminus of colicin Ia to construct colicin Ia-antibody mimetic fusion proteins (Figure 1A-E). The targeting domain in one of them is a mimetic of a mAb (NCBI 2MPA) that recognizes the OmpA of N. meningitides, a member of the OmpA family which shares epitopes with over 600 other microbes, including Gram negatives (E. coli, K. peumoniae, E. cloacae, Y. pestis, V. cholerae) and Gram positives including M. tuberculosis (Mtb) [5- 8,21]. This porin A-specific antimicrobial fusion protein was named pheromonicin-NM (PMC-NM). Interestingly, PMC-NM does not inhibit the growth of certain probiotics, e.g., Bifidobacterium longum and Lactococcus lactis, presumably due to their lack of similar OmpA analogs on their surface (Figure S3A).

Among several tested pheromonicins with relevant antibody mimetics derived from antibodies targeting different epitopes, only the PMC-NM interacted with OmpATb of Mtb (Figure 1F). PMC- NM inhibited the growth of OmpA pathogens as a bactericidal agent (Figure 1G) [17,18]. In contrast, significant differences regarding in vitro activity indicated that neither wild-type colicin Ia, nor reversed PMC-NM with a scramble mimetic, is be able to target Mtb cells (Figure 1G).

The results of two-phases Mtb growth inhibition showed that phase I (6-wk), PMC-NM (1 ug/ml) inhibited the growth of H37Rv cells while wild-type colicin Ia (1 ug/ml) failed; phase II (10-wk), wild- type colicin Ia (2 ug/ml) still did not inhibit the growth of H37Rv cells. Interestingly, in the phase II tube which was inoculated with remains of phase I PMC-NM-treatment culture, no H37Rv cells re-grew, suggesting that PMC-NM was bactericidal (Figure 1H).

A Minimum Inhibitory Concentration (MIC) assay of 39 isolates of MDR-TB (collected by China CDC) showed there were significant differences regarding MIC50 and MIC90 among PMC-NM, wild-type colicin Ia and reversed PMC-NM (Table 1).

Table 1: MIC (ug/ml) of PM-NM and colicin Ia against 39 isolates of MDR-TB.

Above results indicated that only the PMC-NM with constructed NM antibody mimetic with accurate N- to C-terminal sequence could target the Mtb cells.

A MIC assay of several pan-susceptible Mtb strains (Mtb Erdman, Mtb HN878 etc) and 506 clinical isolates of MDR-TB (collected by China CDC from southern/western China with 10% XDR-TB) indicated that the PMC-NM MIC of pan-susceptible Mtb strains were 0.027-0.11 nmol (1.875-3.75 x 10-3 μg/ml) and the MIC50/MIC90 of MDR-TB isolates was 1.14/143 nmol (0.08/10 μg/ml), which were significantly lower than the MIC50/MIC90 of 11 standard anti-TB drugs (Figure1I and Table 2). Considering the differences in molecular weight between PMC-NM (70 kDa) and standard anti-TB agents (≤1 kDa), the inhibitory activity of PMC-NM against pan-susceptible Mtb strains and MDR-TB isolates was approximately 103-5 times greater, on a molar basis, than that of standard anti-TB agents. The inclusion of 10% XDR isolates likely expanded the MIC range of PMC-NM.

Table 2: Killing rate against Mtb cell/human pulmonary cell culture

PMC-NM suppressed the pulmonary TB burdens and altered the clinical outcomes in murine pan-susceptible/MDR-TB infection.

Figure 1: Structure and in vitro bioactivity of PMC-NM.
(A) Structure of the antibody mimetic comprising the V CDR1, V FR2, V CDR3 domains of Fab of 2MPA mAb. (B) 8.3 kbp colicin Ia plasmid used for site-directed mutagenesis of colicin Ia gene to insert the gene encoding the antibody mimH etic. (C)HA domLain diagram of the PMC-NM. (D) The Rev-PMC-NM constructed with scramble mimetic. (E) PMC-NM (70 kDa) comprised about 90% of total SDS eluted protein. Lanes, (1) marker, (2) PMC-NM.

To evaluate whether PMC-NM suppressed the pulmonary TB burdens and altered the clinical outcomes of murine TB infection, we first tested the PMC-NM efficacy against drug-sensitive Mtb Erdman infection model. We subsequently studied PMC-NM efficacy with a mildly drug-resistant Mtb strain infection model using a short- term (30 days) treatment. Female BALB/C mice (six per group) were infected intranasally with 6.8×102 CFU of Mtb Erdman. Twenty-one days post-infection mice were treated with either intraperitoneal injection (i.p.) of saline, PMC-NM (20 or 40 mg/kg/d), or oral delivery of INH (25 mg/kg/d) for 4 weeks. Right lungs were collected for TB CFU determination. Both INH and PMC-NM treatment significantly reduced TB CFU about 102-3 logs compared to that of controls (Figure 2A).

We tested several doses (5, 10 and 20 mg/kg/d, i.p.) of PMC-NM in female BALB/C mice infected with a fatal inoculum of MDR-TB [MDR-06005, PMC-NM (MIC=0.08 μg/ml), INH (MIC > 0.2 μg/ml), levofloxacin (MIC=4 μg/ml), and moxifloxacin (MIC=2 μg/ml), 0.2 ml, 105-6 CFU/ml] given through the tail vein. We found that 10 mg/kg/d of PMC-NM yielded a 75-80% survival rate but 5 mg/kg/d of PMC- NM failed (Figure 2B). Subsequently, we selected 10 mg/kg/d as an in vivo protective dose against MDR-TB infection for further studies.

The PMC-NM molecules were able to selectively accumulate in the pulmonary TB lesions 1 to 12 hrs after a one-time i.p. application (10 mg/kg) (Figure 2C).

PMC-NM treatments significantly reduced TB burdens and infection levels in macaque pan-susceptible Mtb infection [22-26]. Since primate models are most similar clinically and pathologically to human TB infection, cynomolgus macaques were selected to determine in vivo efficacy of PMC-NM. Healthy cynomolgus macaques could tolerate 2-wk PMC-NM treatment with daily intravenous injection (i.v.) as the dose increased from 0.8 to 2 mg/kg/d with no apparent abnormalities.

Sixteen macaques were studied using several regimens to compare 5-wk PMC-NM treatment (i.v. 2 mg/kg/d, n=6) with a saline-treated control group (n=6) and orally dosed INH groups (0.1 mg/kg/d, n=2), or (1.0 mg/kg/d, n=2). Five hundred CFU pan-susceptible H37Rv cells was inoculated into the right caudal lung lobe of each macaque through a bronchoscope. Treatment started at day 24 post -infection. Comparative measurements of TB CFU and pathology scores suggested that PMC-NM treatments significantly reduced TB burdens and pathology score in lung tissues (Figure 2D-E and Figure S1A).

Figure 2: (A) Immunoblotting assay showed that only the PMC-NM, not other PMCs, interacted with extracted OmpATb of Mtb H37Rv (about 114 kDa). Lanes, (1) PMC-NM, (2) PMC- cyanobacteria, (3) PMC-B.dendrobatidis, (4) PMC-HB8627, (5) PMC-N.gonorrhoeae. (B) PMC-NM inhibited the growth of OmpATb pathogens (Mtb H37Rv) as a bactericidal agent. (C) Wild-type colicin Ia could not inhibit but PMC-NM inhibited the growth of H37Rv in the test tube. Phase I (6-wk), all tubes inoculated with H37Rv cells, (a) control, (b) wild-type colicin Ia 1 ug/ml, (c) PMC-NM 1 ug/ml, phase II (17-wk), all tubes inoculated with H37Rv cells, but tube g inoculated with remains of phase I PMC-NM-treated culture. (d) control, (e) spare borate buffer, (f) wild-type colicin Ia 2 ug/ml. (D) The MIC frequency distribution of PMC-NM against measured several drug-sensitive Mtb strains and 506 isolates of MDR-TB/XDR-TB. (E) After 2 hrs incubation with (Ea) INH 1 μg/ml, or (Eb) PMC-NM 2 μg/ml, INH did not show any efficacy on MDR-TB cells but PMC-NM killed all MDR-TB cells. Bar, 10 or 20 μm. (F) Comparing with normal MDR-TB cells (Fa), (Fb) Destroyed MDR-TB cells were either ruptured, or shrinkage as empty cell-ghosts with leakage of cellular contents with PMC-NM treatment (2 μg/ml).

PMC-NM Altered the Clinical Outcomes in Macaque MDR- TB Infection

Pharmacokinetic assays indicated that i.p. application provided a longer circulatory retention contour than that of i.v. application (Figure 3A), therefore, we selected i.p. delivery to yield a longer PMC- NM exposure in pulmonary lesions.

A high virulence MDR-TB isolate [PUMC-94789], PMC-NM (MIC=0.08 μg/ml), INH (MIC=4 μg/ml), RIF (MIC=64 μg/ml), ethambutol (MIC=4 μg/ml) was selected to infect macaques (Table 3). Four-wks of PMC-NM treatment (i.p. 3 mg/kg/d, n=4) was compared to untreated controls (n=4)( Figure S1B). Results indicated that (a) 300 CFU of PUMC-94789 cells created a successful infection model, (b) i.p. PMC- NM was efficacious in this model.

Table 3: Killing rate against Mtb cell/human pulmonary cell culture

Note: Conc.2 : Seed 0.5ml-A549/well at 2×105/ml in 24 well flat-plate. Conc.1 : Seed 0.5ml-A549/well at 1×105/ml in 24 well flat-plate.
Comments:
1. The killing rate as control group, Isoniazid (1ug/ml), reaches 78% on day 3.
2. The killing rate as exp. group, pheromonicin (1ug/ml & 10ug/ml), reaches 33% & 61% respectively on day 3.
3. A549 cells will be apoptotic from on day 3 as critical point under the exp. concentrations.
4. Mtb bacteria die on day 7 with loss of cell host.

Sixteen macaques were infected with 300 CFU of PUMC-94789 cells in the right caudal lung lobe. Infection was confirmed by eyelid tuberculin testing and CT scans 3-wks after inoculation. The animals were randomly allocated into three 22-wk treatment groups: untreated controls (n=4), combined INH/RIF-treated (i.p. 21.8/3.65 μmol/kg/d (3/3 mg/kg/d), n=4, doses adjusted from reported MDR-TB rodent studies) [27,28], and PMC-NM-treated (i.p. 43 nmol/kg/d (3 mg/ kg/d), dose adjusted from previous rodent/macaque doses, n=8). Treatment was initiated day 24 post-infection. Seventy-five per cent of PMC-NM-treated group survived. All untreated and INH/RIF- treated controls and two PMC-NM-treated macaques reached the humane endpoint within 10-17 weeks post-infection. All macaques that reached the humane endpoint suffered from clinical symptoms related to MDR-TB infection confirmed at necropsy (Figure 3B, C and Figure S2A-C). Serial CT scans were performed before the infection, and during the entire test period [29,30]. The whole pulmonary TB lesion volume in each CT scan was the summation of lesions in every 1-mm thick digitally dissected CT image (Figure S2A and S2D). The clinical outcome of each macaque was quantitatively extrapolated as the function of serial variation of TB lesion volumes during the entire test period (Figure 3D-F).

The MDR-TB infection in the macaques underwent different clinical courses with respective treatments (Figure 3B-G and Figure S2A-C), (a) all untreated and INH/RIF-treated macaques were euthanized around 10～17-wk post-infection while TB lesion volumes continuously enlarged with pulmonary TB CFU growth (Figure 3Da, Fa ); (b) two macaques with low bodyweight (≤ 4 kg) were randomly allocated in the PMC-NM group, we assume that poor physiological conditions might be the reason for the apparent failure of PMC-NM. The lung cultures yielded no growth and decreased TB lesion volumes suggesting control of the TB infection (Figure 3Db, Fb); (c) two PMC-NM-treated macaques were euthanized at 47-wk post-infection with relapsed infection which suppressed by a second 18-wk PMC-NM treatment, presumably their doubled lesion volumes compared to the other PMC-NM treated animals at the end of first treatment, facilitated their relapse (Figure 3Ea, Fc); (d) sixty per cent of surviving PMC-treated macaques completed the 52-wk study with no measurable lesion volumes or TB CFU growth but increased their body weight (Figure 3Eb, Fd).

The TB strain which grew in the infected macaque lungs displayed the same resistance profile as the original PUMC-94789 strain (Table 4). The main lesion in untreated primates was acute tuberculosis pneumonia while in INH/RIF-treated primates was granulomas. In contrast, the lesions in PMC-NM-treated primates was significantly less than above groups (Figure 3G,H and Figure S2E).

Figure 3: Probably PMC-NM targeting/killing mechanism.
(A) Antibody mimetic (green) interacted with OmpATb to drag the channel-forming domain (orange) to bind the cell membrane, (B) hydrophobic interaction drove the channel-forming domain to form channel, (C) transmembrane potential opened the channel resulting in the leakage of cellular contents.

Table 4:

The serum biochemical data of untreated and PMC-treated monkeys were compared in reference to normal values (Table 5), The BUN/CRE of PMC-treated were in normal range but that of untreated were upper limit of normal or above ; the GLU and TG values of untreated were low, the CK and LDH of untreated were almost 10 times higher than upper limit of normal indicated there might be some muscle/bone and multi-organ TB lesions. Above significant differences between untreated and PMC-treated serum biochemical data indicated that untreated monkeys had serious multi-organ and metabolic abnormalities due to MDR-TB infection, however, PMC protected monkeys with no apparent toxicity.

Table 5:

Discussion

Our results indicate that daily PMC-NM treatment (i.p. 43 nM/ kg/d for 22-wks), initiated 24 days after fatal MDR-TB infection can significantly reduce MDR-TB lesions in lungs and clinically cure certain cases. To our knowledge, this is the first report of a channel- forming toxin-antibody mimetic fusion protein altering the clinical course in fatal MDR-TB infected rodent and primate models.

Several findings suggested the antibody mimetic domain of PMC-NM molecules targeted the OmpATb appearing on the surface of either Mtb cells, or infected host cells with internalized Mtb cells [5-10,31-35]; (a) immunoblotting data showed only PMC-NM with 28-residue antibody mimetic of 2MPA mAb, not other tested pheromonicins, interacted with OmpATb (Figure 1f), (b) Growth inhibition data demonstrated that without fused 28-residue VHCDR1- VHFR2-VLCDR3 mimetic with accurate N- to C-terminal sequence, wild-type colicin Ia would not be able to recognize OmpATb (Figure 1G), (c)immunofluorescent dyes showed that OmpATb appeared in the infected lung tissues, even traced the outlines of infected cell membrane and organelles (Figure 4B), (d) PMC-NM molecules selectively interacted with OmpATb, then accumulated in the lesion tissues from circulation (Figure 4B), (e) with immuno-labeling electron microscopic assay, paired 5/10 nm colloid gold molecule images revealed the interaction between OmpATb and PMC-NM molecules at infected host cell membrane (Figure 4D,E), and (f) immunoblotting assay showed PMC-NM recognized the membrane precipitates of the infected lungs, as well as OmpATb of Mtb cells, but did not recognize the precipitates of uninfected and PMC-NM-treated lungs (Figure 1F and 4F).

Early OmpATb studies suggested that OmpATb might act on the outer membrane as a monomer pore (38 kDa)(5, 8). In our immunoblotting assay, the OmpATb precipitations of H37Rv were around 114 kDa, if the interaction with the PMC-NM occurred less than 24 hrs after OmpATb preparation was made, however, they were degraded as 38 kDa precipitations, if the interaction with the PMC- NM occurred beyond 48 hrs. These findings suggest that in vivo, at least some part of OmpATb might be in trimmeric form (Figure S3).

Two main domains of PMC-NM are involved in killing Mtb cells, (a) antibody mimetic interacted with OmpATb to drag the channel-forming domain to bind the cell membrane, (b) hydrophobic interaction drove the channel-forming domain to form channel in the cell membrane irreversibly, (c) transmembrane potential opened the channel to induce the leakage of cellular contents resulting in cell death (Figure 4G). Cellular content leakage through the large ion-conductive lumen (9-11 Å) of the colicin channel is a novel mechanism of action against TB cells, as well as infected host cells (9-12,16-19). Either rupture, or shrinkage as empty cell-ghosts occurred in all cells treated with PMC-NM (Figure 4H,I). PMC-NM could therefore target/kill any cell, either prokaryocytes, or eukaryocytes, with lipid bilayer cell membranes, if OmpATb appeared on their surface. Future studies will help to further define the specific mechanism of action of PMC-NM.

Figure 4: In vivo activity of PMC-NM in murine and macaque TB.
(A) Mtb CFU of Mtb Erdman infected mice lung after 4-wk treatment with either saline, INH, or PMC-NM. t test was used to determine significant differences between the PMC-NM and saline groups (p<0.05), INH and saline groups (p<0.05). (B) Cumulative survival of MDR-TB (MDR-06005) infected mice with 4-wk injected intraperitonealy (i.p.) with saline, INH 6, 12 mg/kg/d, or PMC-NM 10, 20 mg/kg/d; or with saline, or PMC-NM 5, 10, 20 mg/kg/d. The numbers of surviving mice at various time points were determined, and Kaplan-Meier analysis was used to determine significant differences between the PMC-NM 10, or 20 mg/ml treatment with other treatments (p<0.05). (C) Immunolabeled PMC-NM molecules (green) selectively accumulated in mice pulmonary MDR-TB lesions at 1 to 12 hrs after one- t ime i.p. application (40x, inlet, optical images). (D) 5-wk i.v. 2 mg/kg/d PMC-NM significantly reduced Mtb H37Rv burdens in macaque lungs. (E) PMC-NM significantly controlled macaque gross TB pathology and lesions.

Figure 5: In vivo activity of PMC-NM against MDR-TB infection in macaque.
(A) Pharmacokinetic assays indicate that i.p. application provided a longer circulatory retention contour. (B, C) Cumulative survival of PMC-NM-treated macaques and controls. The numbers of surviving animals at various time points were determined, and Kaplan-Meier analysis was used to determine the significance of difference between the PMC-NM and control groups (p=0.003) and the non relapsed and relapsed cases (p<0.05). (D) Variations of pulmonary TB lesion volumes with different treatments, (a)Untreated and INH / RIF, n=8, (b) euthanized of PMC-NM, n=2.  (E) Variations of pulmonary TB lesion volumes with PMC-NM treatment, (a) relapsed, n=2, (b) non relapsed, n=4. (F) The clinical outcomes of respective treatments were extrapolated as the function of D and E variations, (a) untreated and INH/RIF treatments; (b) euthanized, (c) relapsed and (d) non relapsed cases of PMC-NM treatment. (G) MDR- TB burdens of the whole lung. Each symbol is one macaque. Macroscopic view of lungs of control (Ha,b), INH/RIF-treated (Hc,d, granulomas, yellow circles) and PMC-NM- treated macaques (He,f). Microscopic view of lungs, (Ia) Miliary TB dissemination with necrotic mineralization in the lung of untreated controls. (Ib) Aggravated granulomas in the lung of INH/RIF-treated controls. Conversely, the lung appearance of PMC-NM treated animals was almost normal at the end of 22-wk treatment (Ic), or at the end of 52-wk test (Id). (40x, HE staining)

Figure 6: Probably PMC-NM targeting/killing mechanism.
(A) Untreated control. (200x) (B) OmpATb appeared on cell membranes and organelles of infected host cells (insets).(200x) (C) PMC-NM molecules accumulated on the surface of infected host cells through circulation. (200x) (D,E) Mtb and Infected host cells were probed with anti-OmpA and anti-PMC-NM Abs with 5 and 10 nm colloid gold molecules (grey and black arrows, respectively). Paired gold molecules revealed the OmpATb/PMC-NM interaction around the cell membranes (blue arrows). Bar, 0.2–1 μm. (inset, the location of higher resolution images). (F) Immunoblotting assay showed that PMC-NM interacted with the membrane precipitates of infected macaque lung, as well as OmpATb of Mtb cells, but interacted nothing with the precipitates of uninfected and PMC-NM-treated lungs. Lanes, (1) Mtb H37Rv, (2) infected lung, (3) uninfected lung, (4) PMC-NM-treated infected lung.

Comparing with activities of tested standard anti-TB agents, MIC of pan-susceptible Mtb strains/MDR-TB isolates and in vivo efficacy indicated that PMC-NM presents a new mechanism of action. Cellular content leakage induced by PMC-NM channels is much more robust, as compared to biochemical activity of standard anti-TB agents which is more easily interrupted through mutagenesis. Present resistance mechanisms of MDR-TB, such as interfering with the binding of drug to target protein, or reducing drug-activating enzymes, presumably were unable to block the activation of PMC-NM channels in the TB cell membrane.

Notably, the MICs of PMC-NM against MDR and XDR Mtb strains were commonly higher than that of against drug-sensitive Mbt strains. The following factors might explain the variance of PMC- NM inhibition concentrations across various clinical and lab strains of Mtb, (a) mutant or other structural alterations of Mtb surface, (b) differential expression of OmpATb by these Mtb strains, or the differential presentation of OmpATb on the surface of infected host cells and (c) functional alteration of antibody mimetic domain of PMC-NM. We will continue related investigation in future studies.

Normal blood biochemical parameters, and lack of weight loss or abnormal behavior indicated that treatment with PMC-NM for 22-wks did not induce evident toxicity (Table 5). PMC-NM in vivo bactericidal efficacy against MDR-TB infection indicated that PMC- NM appears to act in the host circulation with full bioactivity.

Table 6:

Additional studies will be necessary to better understand the potential therapeutic efficacy of PMC-NM in multi-drug regimens in combination with anti-TB chemotherapy.

Our data supports the further development of this biological agent and future use with an optimized background regimen against MDR- TB infection in human trials.

Acknowledgment

This work was supported by National Science and Technology Major Projects of New Drugs 2012ZX09103301-024 and 2015ZX09102007-014, National High Technology Research and Development Program of China 2011AA10A214 of Ministry of Science and Technology; Beijing Municipal Science & Technology Commission Z131100002713010 and Z161100000116016 to X-Q.Q.

We would like to acknowledge the help and scientific critique of H. Li, G. Zeng and CY. Jiang during the preparation of this manuscript. We would also like to acknowledge the help of HL Ma, SB Liang, TF Liu, WC Wang, JY Tang, F Meng, XC Li and BG Yuan in PMC-NM preparation and animal care.

Author Distribution

X-Q.Q., K-F.C., Z-H.X., X-F.Z., Y. P., C-Y.T., C.S., R-Q.L, and

M.C. prepared antibody mimetics and pheromonicin-NM molecules, measured in vitro and in vivo activity; X-D.H., and G-K.O. did CT assays and analysis; X-Q.Q., M.C., S.K., Y-L. Z., and Y.W. designed and organized the study and manuscript.

Competing Interests

All authors declare that they have no conflicts of interest.

Summary

Antibody mimetic interaction with OmpATb initiated the channel formation in target cell membrane, then formed channel led cellular content leakage is a novel mechanism of action against MDR-Tb cells. PMC-NM demonstrated high efficacy against pan-susceptible and MDR-TB strains, suppressed the pulmonary TB burdens in murine/ macaque TB models and altered the clinical outcomes in macaque MDR-TB infection. Above in vivo efficacy indicated that PMC-NM appears to act in the host circulation with full bioactivity and no evident toxicity. Our data supports the further development of this biological agent and future use against MDR-TB infection in human trials.

Materials & Correspondence Data and material availability: All isolates of MDR-TB are available from China Centers for Disease Control, Beijing (pangyu@chinatb.org). The PMC-NM source and methods are available from Lab. of Biomembrane & Membrane Proteins, West China Hospital, Sichuan University (491607484@ qq.com). All CT data are available from Dept. of Radiology, 309 Hospital, Beijing (hxd83405107@163.com).

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Treatment of Genital Warts in Pregnant Women

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DOI: 10.31038/AWHC.2024752

Abstract

There are various treatments available for managing genital warts in pregnant women, but this does not mean that there is a definitive treatment protocol. We conducted a review to gain a better understanding of the available treatments for genital warts during pregnancy. We used Google Scholar, PubMed, and Web of Science databases to search for articles on this topic. To date, the most effective treatment is still unknown. Many indicators, including patient satisfaction and physiological conditions, are effective in determining treatment. Cryotherapy, laser therapy, and Trichloracetic acid 80–90% solution are among the safe treatments with the least side effects. Given that the likelihood of genital warts recurring increases due to changes in the body’s immune system during pregnancy especially in younger pregnant women, more research is needed on this subject. We used the keywords pregnancy, genital warts, HPV, cryotherapy, laser therapy, electrocautery, surgery, topical treatments, and safe treatments in pregnancy in the article search process.

Keywords

Pregnancy, genital warts, HPV, Cryotherapy, Laser therapy, Topical treatments, Common and safe treatments

Introduction

Human papillomavirus (HPV) poses a significant public health challenge, particularly due to its role in various anogenital conditions, including condyloma acuminata. This virus consists of over 100 different types, which are categorized into low-risk and high-risk groups based on their potential to cause cancer. Low-risk types, especially HPV 6 and 11, are primarily responsible for benign lesions such as genital warts, while high-risk types, including HPV 16 and 18, are associated with cervical cancer and other anogenital malignancie.

The global prevalence of HPV infection is estimated to be between 9% and 13%, with a notably higher incidence observed among sexually active individuals aged 20 to 39 years. Genital warts, largely caused by HPV, are a considerable public health issue due to their widespread occurrence and related complications. The often asymptomatic nature of HPV infections can lead to underdiagnosis, particularly among partners of infected individuals. Research assessing the prevalence of HPV in spouses of men with genital warts revealed that many of these women were infected despite appearing healthy during clinical evaluations, highlighting the inadequacies of conventional diagnostic techniques [1-3].

Pregnancy creates a distinct environment for HPV infections, as physiological changes and a mildly immunocompromised state can affect the virus’s behaviour. Evidence suggests that the rate of HPV infections increases during pregnancy, particularly in the third trimester [2,4]. Effective treatment options for genital warts are crucial, especially for vulnerable populations such as pregnant women. Among the most commonly employed treatments are cryotherapy and trichloroacetic acid (TCA), each having distinct efficacy and safety profiles. A randomized controlled trial comparing these two treatment modalities indicated that while TCA had a higher cure rate, both treatments were effective and well-tolerated by patients. This emphasizes the necessity for tailored treatment strategies that take into account patient preferences and the specific clinical situation, especially when managing genital warts related to HPV infection [5].

Managing genital warts in pregnant women presents several challenges due to the potential risks associated with various treatment options. Traditional approaches, such as surgical excision, may pose risks to both the mother and the fetus. As a result, less invasive alternatives like cryotherapy have become preferred first-line treatments because of their safety and effectiveness [2,6]. Additionally, the possibility of vertical transmission of HPV during childbirth raises concerns about neonatal health outcomes, including the risk of juvenile laryngeal papillomatosis [2]. Given the complexities involved in treating HPV-related genital warts during pregnancy, it is essential to investigate optimal management strategies for this demographic. This study aims to assess the efficacy and safety of various treatment modalities for genital warts associated with HPV in pregnant women, with a focus on minimizing risks to both maternal and neonatal health. Through this research, we aspire to improve clinical practices and contribute to the broader understanding of managing HPV-related conditions in pregnant patients.

Methods

A review of the keywords of scientific journal articles on the management of genital warts in pregnant women showed that the important issue in the management of this disease in pregnancy is the presence of the least side effects of the selected treatment on the mother and fetus, and common but teratogenic treatments are completely excluded from the treatment strategies. We tried to extracted and summarize the studies related to the title in order to provide useful information to the readers. In writing this review, the scientific databases PubMed, Scopus, Google Scholar and Web of Science were used.

HPV in Pregnancy

Human papillomavirus (HPV) infection is among the most prevalent sexually transmitted infections in both young individuals and adults. It often presents without symptoms, can remain dormant for months, and may not be detectable through routine clinical examinations. Despite its asymptomatic nature, HPV infection can progress to cancer, contributing significantly to morbidity and mortality worldwide [3]. Although HPV infections are generally transient and self-limiting, they can persist in immunocompromised individuals, and pregnancy is considered a state of immunosuppression Pregnant women are at a higher risk of HPV infection due to their altered immunological and endocrinological status [7]. In a review study with 13,640 pregnant women, the rate of HPV infection was higher than that of age-matched non-pregnant women, especially in those under 25 years of age. HPV-16 was the most common observed type [2]. This prevalence is two times higher in pregnant women compared to non-pregnant women. Furthermore, there is a potential risk of vertical transmission of HPV from the mother to the fetus during pregnancy or delivery. HPV cervical infections are also detected more frequently during pregnancy (15.53%) than in the non-pregnant population (12.6%). A systematics review that analyzed 14 articles involving a total of 7008 women demonstrated the significant association was identified between HPV infection and preterm delivery A comparative study showed a higher rate of HPV infection in women with abnormal course of the first trimester of pregnancy Compared to women with a normal pregnancy period. Intrauterine HPV infections are also possible, as HPV DNA has been detected in the placenta, amniotic fluid, and fetal membranes. Transmission to the fetus can occur through ascending infection from the vagina and cervix or via hematogenous spread. Studies further indicate that HPV can infect trophoblastic cells, potentially impacting the intrauterine environment and influencing pregnancy outcomes [7-9].

The Prevalence of HPV Infection in Pregnant Women

Pregnancy-related hormonal changes, including mild immunosuppression and elevated steroid levels, may further promote HPV replication. These physiological changes often lead to the proliferation of condyloma acuminata, resulting in larger lesions [10-12]. While some studies suggest pregnancy as a risk factor for HPV infection, findings remain inconsistent [2,13-15].

A meta-analysis by Liu et al reported a higher prevalence of HPV in pregnant women (16.82%) compared to non-pregnant women (12.2%) [2]. More recently, Ardekani and collogues, found that nearly one-third of pregnant women globally test positive for HPV based on cervico-vaginal samples. HPV infection during pregnancy has been associated with significant maternal and fetal morbidity and mortality [16,17]. Studies link HPV to placental abnormalities and adverse outcomes, including spontaneous abortion, preterm birth, premature rupture of membranes, intrauterine growth restriction, fetal death, and low birth weight [11,18]. Regarding HPV prevalence across pregnancy trimesters, results have varied. While Ardekani et al, reported the highest prevalence in the second trimester [16]. Liu et al, found the lowest prevalence during this period [2]. Ambühl et al, observed a declining trend from the first to the third trimester [14]. Globally, HPV types 16 and 18 are the most prevalent across all sampling sites [16]. Due to their oncogenic potential, these types are linked to pregnancy complications and future cancer risks, emphasizing the need for further research and preventive measures [19,20].

Treatment Strategy in Pregnant Women

Pregnancy creates a situation in which genital warts may recur and grow more rapidly in pregnant women than in non-pregnant women. Growth is most rapid between weeks 12 and 14 of pregnancy, and the lesions are larger in size. The severity of these processes is greater in new warts that develop during pregnancy. Changes such as decreased cellular immunity and increased blood vessel number, which consequently leads to increased blood flow in the genital area, increase human papillomavirus activity. Among the features of warts that develop during pregnancy, could mention their fragility of their structure, which leads to itching and bleeding [21,22]. Warts that become exceptionally large can cause labor dystonia or heavy bleeding by blocking the birth canal, and in such cases, a cesarean section is recommended for patients. There is a risk of human papillomavirus transmission from mother to fetus during childbirth. Infants infected with the virus may develop lesions on the conjunctiva, mouth, and/or genitals. [22-24]. Prenatal transmission of HPV types 6 and 11 from mother to fetus can rarely cause juvenile laryngeal papillomatosis (JLP). These clinical observations make effective management of genital warts during pregnancy essential. Our goal in this review is to provide a summary of approved and unapproved treatments for genital warts during pregnancy.

Cryotherapy

Cryotherapy is a therapeutic process in which liquid nitrogen causes tissue freezing and ultimately necrosis. Cryotherapy is one of the main treatments for genital warts, which, in addition to causing tissue necrosis, can stimulate specific immune responses against the remaining lesion tissue, resulting in the immunomodulatory function of T lymphocytes [25,26]. One of the unique features of cryotherapy is that it is safe during pregnancy. In addition, it is a simple and inexpensive treatment that rarely causes scarring or pigmentation in patients’ skin [6]. The liquid nitrogen used in cryotherapy causes inflammation as it destroys the wart tissue, which subsides after a short period of time [27]. In cryotherapy, the base of the lesions and 1 to 2 millimeters of surrounding normal tissue are frozen. This treatment is continued every 2 weeks until the lesions are completely gone [27-29]. Although there have been reports of swelling, discharge, erythema, and pain in patients, all patients were able to complete the treatment. In the study by Bergman et al., cryotherapy resulted in two preterm deliveries in 28 pregnant women (7.1%) [30,31] and in the study by Matsunaga et al., in 51 pregnant women, it resulted in five preterm deliveries (9.8%). Cryotherapy is a suitable treatment modality in non-pregnant patients, and results in satisfactory results, but it should be used with caution in pregnant women. Despite the weak evidence of the side effects mentioned, cryotherapy is considered a safe treatment for genital warts during pregnancy [28,32].

Laser Therapy

The advantages of using laser therapy include high precision of operation, reduced bleeding through vessel sealing and reduced scar surface. In addition, this treatment method has the ability to cause necrosis in a specific area. This ability leads to reduced inflammation, edema and infection . In a study conducted by Kryger and Baggesen, 15 pregnant women underwent laser therapy for genital warts. Except for one pregnant woman who experienced “symptoms of preterm labor for a few days,” the rest responded to the treatment without any side effects [33,34]. The use of CO2 lasers and Nd-YAG lasers is known as effective treatment methods for removing genital warts. This treatment method reduces the risk of recurrence and complications during childbirth and prevents infection of the fetus [30,35,36]. Overall, various studies showed that the use of lasers for the removal of vascular lesions of the skin, pigmented lesions and treatment of genital warts is a safe and effective method [37].

Electrocautery and Surgery

Surgery is a mechanical procedure using a scalpel or scissors that allows for direct removal of the genital wart. The use of electrical energy during surgery is called electrosurgery, which is a more advanced method of lesion removal [28]. In a study conducted by Duus et al., it was shown that the clearance, recurrence, and postoperative side effects including pain, healing time, and scar formation of surgical treatments were similar to laser therapy [38]. One advantage of these methods is the possibility of removing all lesions in one session and creating the opportunity for pathological evaluation [30]. In addition, they are considered a suitable treatment option during pregnancy [39].

Photodynamic Therapy

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) method clears a very large volume of lesions and is well tolerated by the patient. This method is a unique treatment in pregnant women with genital warts with minimal adverse effects on the mother and fetus [40]. Other studies have shown that photodynamic therapy, in addition to helping to better clear the lesion, reduces the recurrence rate more than other treatment methods. However, further studies are recommended to ensure fewer side effects and the safety of this treatment in pregnant women [30].

5-Fluorouracil

In addition to inhibiting DNA synthesis, 5-fluorouracil can block thymidylate synthase, thereby inducing apoptosis [41]. It is available commercially as a 5% cream. Pain, burning, inflammation, and ulceration are known side effects of 5-fluorouracil therapy [42]. Studies have shown that 5-fluorouracil therapy should not be used during pregnancy [30,41].

Interferon

Interferon heals virus-infected cells by strengthening the immune system. Interferon can reduce the risk of relapse by treating virus-infected cells. In addition, it is also used to treat lesions that are visible to the naked eye [43,44]. Interferon is contraindicated during pregnancy [30]. Because interferon therapy is expensive, and because it is controversial in the treatment of genital warts, it is preferred for treatment-resistant cases [45]. In refractory non-pregnant patients, interferon may be used as an adjunct to other treatment strategies, such as surgical and ablative therapies. Overall, the evaluations suggest that interferon is contraindicated in pregnancy [41].

Podophyllotoxin

Topical podophyllotoxin, which is prescribed as one of the first-line medications for the treatment of genital warts, is contraindicated during pregnancy, All medications that use this compound in their structure are contraindicated during pregnancy due to its antimitotic properties, which are considered a toxic compound for the fetus [46-48]. No teratogenic effects have been observed following topical administration in animal studies, but the use of this drug during pregnancy is limited due to the limitations in extrapolating animal data to humans. The lack of teratogenic effects has also been demonstrated at doses up to 5 times the maximum recommended human dose [49-51]. Studies have shown that doses much higher than the maximum recommended human dose, approximately 19 times, administered intraperitoneally to pregnant rats resulted in fetal toxicity. However, topical application of podophyllotoxin has shown negligible systemic absorption. Avoiding topical treatments for genital warts with Topical podophyllotoxin is recommended during pregnancy [52-54].

Sinecathecins

Treatment of genital warts with syncatechin resulted in the occurrence of the side effects of urethral stricture, urinary tract irritation, genital herpes simplex, vulvitis, hypersensitivity, skin pruritus, pyelonephritis, application site reactions, phimosis, and inguinal lymphadenitis in 5% of patients, which led to discontinuation of further treatment. Sinecatechins is FDA Pregnancy Category C According to the FDA, Sinecatechins is a Category C drug and is contraindicated during pregnancy [55].

Retinoids

Retinoids are contraindicated in pregnant women and women attempting to conceive because of their teratogenic effects. Retinoid therapy during pregnancy has been associated with skeletal abnormalities that result from long-term chronic toxicity. In most cases, these symptoms correspond to the symptoms of diffuse idiopathic hyperostosis syndrome [56].

Imiquimod 5% Cream

Imiquimod was first approved by the U.S. Food and Drug Administration (FDA) in 1997 as a treatment option for genital warts. Randomized, double-blind, placebo-controlled trials have confirmed the effectiveness of imiquimod. It is a member of a class of immune response modifiers that is well tolerated. The U.S. Food and Drug Administration does not recommend the use of imiquimod during pregnancy and has classified it as a category B drug. It should only be used as a last resort when the benefits outweigh the risks [57-59].

Trichloracetic acid 80–90% Solution

The therapeutic effect of trichloroacetic acid on genital warts is by chemical coagulation of the proteins of the wart cells, which leads to corrosion of the skin and mucous membranes. These processes ultimately lead to tissue necrosis. Because trichloroacetic acid is not absorbed through the skin and mucous membranes, it is considered a safe treatment for pregnant women. However, the effectiveness of this compound in different populations requires further study [27,30]. Studies have shown the positive effect of administering trichloroacetic acid in combination with laser therapy in pregnant women. In another study, treatment with trichloroacetic acid resulted in complete clearance of the wart lesions in 97% of women, but premature rupture of membranes in one patient at 35 weeks of gestation and acute pyelonephritis in another pregnant patient were side effects of this type of treatment. However, it is not definitively determined whether these adverse effects were side effects of the drug [60]. Despite the widespread use of trichloroacetic acid in clinical practice, this drug should be prescribed with caution [47].

Topical and Intralesional Immunotherapy

The use of immunotherapy in the treatment of genital warts has been associated with good efficacy. The advantages of this method include high safety, low recurrence rate, and clearance of untreated long-term warts. Bayoumy et al., showed showed that the use of immunotherapy in 40 pregnant women resulted in improvement in 85% of patients. Among them, 47.5% showed complete clearance with minimal side effects. This treatment leads to increased serum interleukin-12 (IL-12) and the production of Th1 cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), IL-8 through recruitment of antigen-presenting cells. It also helps to clear warts through a general response that includes the secretion of interferon (IFN)-gamma and TNF-alpha. This treatment method is considered safe for use in children and pregnant women [61-63].

Conclusion

Given that genital warts are among one of the common sexually transmitted diseases, and that pregnant mothers are at risk of contracting this disease, it seems appropriate to pay attention to these patients and choose the appropriate treatment for them. During pregnancy, the exacerbation of genital warts and the risk of transmitting the disease to the fetus must be considered. Another important point to keep in mind is the limitations of available treatments that can be chosen for mothers during pregnancy. Cryotherapy, TCA application, Topical and intralesional immunotherapy, as well as laser and surgical treatments, are among the treatments that can be usable and safe in during pregnancy.

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Determination of Awareness and Knowledge Levels of Men Between the Ages of 18-45 About HPV and HPV Vaccine

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DOI: 10.31038/AWHC.2024751

Abstract

This study was designed to determine the awareness and knowledge levels of male patients about HPV and the HPV vaccine. This study was planned as a single-center, descriptive and cross-sectional study. This cross-sectional study included 255 patients who presented to the Family Medicine Polyclinic at Tekirdağ Namık Kemal University Hospital. Data were collected between 01.07.2023 and 01.12.2023 with a face-to-face survey form. The content of the survey form included questions about the participants, a survey assessing awareness and knowledge regarding HPV and the HPV vaccine, which we prepared by scanning the literature and whose content validity index was 0.98. The data obtained were evaluated in the 152 (59.6%) of the men stated that they did not have information about HPV, 250 (98.0%) stated that they had not received the HPV vaccine, 102 (40.0%) stated that they did not consider getting the HPV vaccine, and 144 (56,5%) stated they thought it was difficult to access the HPV vaccine. The median (min-max) score of the men in the study group from the survey was 24.0 (6.0-32.0) and the mean (SD) was 23.20. The findings of this study showed that more than half of the men had not heard of the HPV vaccine, almost all of the men had not received the HPV vaccine, and the majority thought it was difficult to access the vaccine. However, men’s general HPV knowledge was average and their knowledge of the current HPV vaccination program was low. In addition, men’s awareness and knowledge survey scores about HPV and the HPV vaccine were at an average level, and HPV knowledge affected men’s attitudes and behaviors. It may be recommended to take initiatives to increase men’s knowledge about general HPV and the HPV vaccine.

Keywords

Man, HPV, HPV vaccine, Knowledge level, Awareness

Introduction

Human Papilloma Virus (HPV) is a DNA virus belonging to the Papillomavirinae family. HPV infects the basal epithelial layer cells of human skin and mucosal surfaces. This virus is a non-enveloped, double-stranded circular DNA virus. The genome of HPV is approximately 8 kilobase pairs long. The replication cycle of HPV is a very slow process. There can be a long period between the onset of HPV infection and the appearance of symptoms, and this period can vary from person to person. It can take weeks or even months from the moment the infection begins to appear for symptoms to appear. HPV can be transmitted directly from cracks in the skin and mucosa, as well as during sexual intercourse. In addition, HPV can be transmitted indirectly from contaminated surfaces (fomites) of shared bathroom floors, towels, clothing or personal belongings, and from the mother to the newborn during birth through the infected birth canal [1-3]. HPV is a common sexually transmitted infection worldwide. There are many different types of HPV, and most types of the virus do not cause any symptoms or health problems in the infected person; however, a small percentage of infections caused by certain types of HPV can persist and cause clinically significant disease. HPV types are classified according to their epidemiological association with cancer. Low-risk HPV types can also cause genital warts and a rare condition called recurrent respiratory papillomatosis. High-risk HPV types are associated with a variety of cancers, including cervical, anal, penile, vaginal, vulvar, and head and neck. Different vaccines have been developed for HPV vaccination, and there are currently three vaccines: bivalent (HPV types 16 and 18), quadrivalent (HPV types 6, 11, 16, and 18), and nonavalent (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58). Each HPV vaccine has been shown to be safe and effective against the HPV strains involved [3-6]. HPV vaccines are used as part of national vaccination programs in the United States, Australia, and many European countries. The fact that HPV vaccines are not included in national vaccination programs and are not covered by health insurance affects access to the vaccine and vaccination rates. Parental knowledge, mentality, and income levels affect whether HPV vaccines are widely used. Increasing resistance to vaccination can make it difficult for vaccines to be accepted and implemented in society [5-7]. HPV research generally focuses on understanding the development of cervical cancer and precancerous lesions, as well as how to prevent and control these conditions. This research focuses particularly on women’s health and the prevention of cervical cancer. The administration of HPV vaccines to women has been seen as an effective strategy for preventing cervical cancer. Recent studies have shown that HPV-related cancers pose a significant burden not only to women but also to men. In addition to HPV being linked to cervical cancer in women, HPV infections in areas such as the anus, oral cavity, and oropharynx pose a serious cancer risk in men [8-10]. HPV infections have now become preventable thanks to HPV vaccines. Countries can increase cancer prevention by expanding such vaccines through vaccination programs [1-4]. The aim of this study was to assess the knowledge and behaviors of men aged 18-45 about HPV and the HPV vaccine.

Material and Methods

This study is a cross-sectional descriptive survey. In order to evaluate the level of knowledge and behavior about HPV and HPV vaccine among male patients aged 18-45 who applied to Tekirdağ Namık Kemal University Hospital Family Medicine Outpatient Clinic between July 1, 2023 and December 1, 2023, the Adult Vaccination Knowledge Level Survey consisting of 16 questions was applied on a voluntary basis. Data from a total of 255 participants were analyzed. Before starting the study, approval was obtained from the Namık Kemal University Faculty of Medicine Ethics Committee (protocol no: 223.129.06.15).

Criteria for Including Participants in the Study

  • Being between the ages of 18-45
  • Having applied to Tekirdağ Namık Kemal University Hospital Family Medicine Polyclinic center
  • Being male
  • Having mental competence
  • Being a volunteer

Criteria for Exclusion of Volunteers from the Study

  • Being under the age of 18 and over the age of 45,
  • Not having mental competence
  • Being a woman

A questionnaire form prepared by the researcher based on literature review at the time of application, which questioned the sociodemographic data of the patients, their knowledge level and behaviors about HPV and HPV vaccine, was filled out by the volunteers.

Validity and Reliability of the Awareness and Knowledge Questionnaire on HPV and HPV Vaccine

A total of 16 items were created to measure awareness and knowledge levels on HPV and HPV vaccine in line with the literature. The suitability and comprehensibility of each scale item was assessed by 10 experts (1 internal medicine, 4 family physicians, 1 emergency, 1 public health, 1 infection, 1 dermatology and 1 gynecology and obstetrics). The experts were asked to evaluate the items in three groups as “important”, “useful but insufficient”, and “unnecessary”. The scope validity rates of the scale ranged from 0.8 to 1.0 and the scope validity index was 0.98.

The Adult Vaccination Knowledge Level Questionnaire, which was created to assess the knowledge level, consists of 16 questions. Factor analysis was used for structural validity. It was performed using Principal Component Analysis (PCA) with Varimax rotation. PCA generally tests the psychometric qualities of structured questionnaires and is a frequently used multivariate statistical technique and is used to determine the structure . The reliability levels expressed by the Cronbach alpha coefficient range were taken into account in the study. For the reliability analyses of the scale, it was calculated again according to the Cronbach alpha coefficient and item total correlation. In the factor analysis, Kaiser-Meyer-Olkin: 0.875, Barlett’s test result was p<0.001. Factor loadings below 0.30 varied between 0.302-0.812, and it was determined that the items collected in a single sub-domain explained 31.63% of the total variance. Cronbach alpha value was determined as 0.814. The item total correlation values ​​for the items in the questionnaire varied between 0.201-0.714. For confirmatory factor analysis, goodness of fit statistic (GFI), adjusted goodness of fit statistic (AGFI), CFI (Comparative fit index), Normed fit index (NFI), Incremental Fit Index (IFI), Relative Fit Index (RFI), Parsimony Fit Index (PNFI), Root Mean Square Error of Approximation (RMSEA) and Standardized Root Mean Square Residual (SRMR) indices were calculated. For the acceptability levels of fit indices, CFI, NFI, IFI and AGFI ≥0.90, RMSEA <0.08 and SRMR ≤0.10 were taken as criteria. As a result of confirmatory factor analysis, no incompatible values ​​were detected for each item for CFI (0.89), NFI (0.81), IFI (0.89), RFI (0.76) and SRMR (0.06). The values ​​were in appropriate ranges. When the fit indices of the model obtained with CFA were examined, it was seen that the X2/df (2.08) value was less than 3, but the CFI (0.89), RFI (0.76), GFI (0.90), AGFI (0.87) and SRMR (0.06) values were at an acceptable level. As a result of the confirmatory factor analysis, these fit indices showed that the model had a good fit [11-14].

The correct answer given to the awareness and knowledge survey items regarding HPV and HPV vaccine was calculated as “2”, the “I don’t know” answer as “1” and the wrong answer as “0”. It was accepted that the awareness and knowledge level increased as the score obtained from the survey increased.

Statistical Analysis

The obtained data were evaluated in the Statistical Package for Social Sciences (SPSS) 25.0 statistical package program. Descriptive statistics were given as mean, standard deviation, median for numerical variables, and number and percentage for categorical variables. In the evaluation of validity and reliability, Exploratory Factor Analysis was applied and factor loadings, item total correlation values, and Cronbach alpha values ​​were calculated for the items. Confirmatory factor analysis was performed using the R studio package program. The conformity of the data to normal distribution was assessed using the Kolmogorov-Smirnov test. Differences between two groups in terms of continuous variables that were not normally distributed were analyzed using the Mann Whitney U test, and differences between multiple groups were analyzed using the Kruskal Wallis test. The results were evaluated at a 95% confidence interval and p<0.05 values ​​were considered significant.

Results

The mean age of the participants was 29.04 ± 6.62 years. 156 (61.2%) of the participants were in the 21-30 age group and 174 (68.2%) were university graduates. 148 (58.0%) of the participants were single, 158 (62.0%) were employed, 73 (28.6) were healthcare workers, and 181 (71.0%) lived in the city center or a metropolitan city. 225 (88.2%) of the participants had no chronic disease and (98.8%) stated that they were heterosexual (Table 1). Of the men, 152 (59.6%) reported that they had not received any information about HPV, 250 (98.0%) had not received the HPV vaccine, 102 (40.0%) did not consider receiving the HPV vaccine, and 144 (56.5%) thought that it was difficult to obtain the HPV vaccine.

Table 1: Distribution of participants according to sociodemographic characteristics and some variables related to HPV.

 

n

%
n

255

100
Age group (year)    
18-20

11

4.3
21-25

82

32.2
25-30

74

29
31-35

51

20
36-40

16

6.3
41-45

21

8.2
Marital status    
Single

148

58
Married

102

40
Widow

5

2
Eğitim düzeyi    
Primary – secondary school

13

5.1
High school

23

9
College

174

68.2
Masters

45

17.7
Occupation    
Working

158

62
Not working

21

8.2
Student

76

29.8
Health worker

73

28.6
Place where lived during most of life
City center

181

71
District

58

22.7
Village

16

6.3
Income level
Income is less than expenses

84

32.9
Income is equal to expenses

102

40
Income is more than expenses

69

27.1
Has children

78

30.6
Smoking

96

37.6
Comorbidity

30

11.8
Sexual orientation
Heterosexual

252

98.8
Homosexual

3

1.2
Given information about HPV vaccine

103

40.4
HPV vaccination status    
Not vaccined

250

98

Gardasil 4/2 dose

 3

1.2
Gardasil 9/2 dose

2

0.8
Considering to be vaccined for HPV    
Yes

55

21.6
No

102

40
Undecided

98

38.4
Considers hard to reach HPV vaccine

111

43.5

184 (72.2%) of the men in the study group thought that HPV was a very common disease in humans. The item with the highest number of correct answers among men (82.7%) was “Having more than one sexual partner increases the risk of HPV transmission.”, while the item with the lowest number of correct answers (8.6%) was “How many types of HPV vaccines are there?” (Table 2).

Table 2: Distribution of responses to the participants’ awareness and knowledge survey regarding HPV and HPV vaccine.

 

n

%
1. HPV is a virus that is quite common in humans.    
Yes (C)

184

72.2
No

71

27.8
2. How many types of HPV are there?    
2

2

0.7
4

10

3.9
9

17

6.7
More than 200 (C)

93

36.5
I don’t know

133

52.2
3. HPV can often be found in a person without any symptoms or signs.    
Yes (C)

153

60
No

19

7.5
I don’t know

83

32.5
4. HPV does not cause disease anywhere on the body except the genital areas.    
Yes

21

8.2
No (C)

145

56.9
I don’t know

89

34.9
5. HPV only causes disease in women.    
Yes

5

2
No (C)

178

69.8
I don’t know

72

28.2
6. HPV can cause mouth, pharynx, anus and penis cancer and genital warts in men.    
Yes (C)

172

67.5
No

5

2
I don’t know

78

30.5
7. HPV is transmitted only through genital contact.    
Yes

59

23.1
No (C)

119

46.7
I don’t know

77

30.2
8. Using a condom reduces the risk of HPV infection.
Yes (C)

193

75.7
No

13

5.1
I don’t know

49

19.2
9. Having more than one sexual partner increases the risk of contracting HPV.
Yes (C)

211

82.7
No

3

1.2
I don’t know

41

16.1
10. Are there any medications that can be used against HPV?
Yes (C)

125

49
No

36

14.1
I don’t know

94

36.9
11. How many types of HPV vaccines are there?
1

8

3.1
2

48

18.8
3 (C)

22

8.6
4

18

7.1
I don’t know

159

62.4
12. What is the best age for the first HPV vaccine?
9 (C)

83

32.5
11

22

8.7
13

28

11
18

114

44.7
45

8

3.1
13. How many doses of HPV vaccine given after the age of 18 provide immunity?
1

9

3.5
2

35

13.8
3 (C)

51

20
I don’t know

160

62.7
14. HPV vaccine has side effects that can cause health problems.
Yes

48

18.8
No (C)

61

23.9
I don’t know

146

57.3
15. HPV vaccine is only administered to women
Yes

10

3.9
No (C)

159

62.4
I don’t know

86

33.7
16. Do you think that HPV vaccination should be given to men?
Yes (C)

201

78.8
No

54

21.2
17. HPV vaccine can be administered to men between the ages of 18-45
Yes (C)

146

57.2
No

15

5.9
I don’t know

94

36.9
18. HPV vaccination in men can contribute to the elimination of HPV (having no patients in the community).
Yes (C)

146

57.3
No

12

4.7
I don’t know

97

38

(C): Correct answer.

In the factor analysis for the awareness and knowledge survey about HPV and HPV vaccine, Kaiser-Meyer-Olkin: 0.875, Barlett’s test result was p<0.001. Two items in the survey (Item 13: 0.021 and Item 14: 0.038) were removed from the survey because their factor loadings were below 0.30. The factor loadings ranged between 0.302 and 0.812, and it was determined that the items collected in a single sub-domain explained 31.63% of the total variance. Cronbach’s alpha value was determined as 0.814. The item-total correlation values ​​for the items in the survey ranged between 0.201 and 0.714 . In addition, according to the confirmatory factor analysis results of the awareness and knowledge scale regarding HPV and HPV vaccine, the x2/dF value was determined as 2.08, AGFI as 0.87, RMSEA as 0.07, SRMR as 0.06, and GFI as 0.90. The path diagram showing the model structure and standard regression coefficients is shown in Figure 1. The median (min-max) score of the men in the study group from the survey was 24.0 (6.0-32.0) and the mean (SD) was found to be 23.20. In the study group, those in the 26-30 age group were found to have higher knowledge and awareness scores than those in the 31-35 age group (p=0.032). Singles had higher scores than married individuals (p=0.014). In terms of educational status, those with primary school education had lower scores than those with university and master’s degrees, those with high school education had lower scores than those with university and master’s degrees, and those with university level education had lower scores than those with master’s degrees (p<0.001). Those who were health workers had higher scores than those who were not (p<0.001). Those whose income was equal to expenses had lower scores than those whose income was greater than expenses (p=0.012). Those who did not have children were found to have higher scores than those who did (p=0.002). Those who had information about HPV vaccination had higher scores than those who had not (p<0.001). Those who considered getting HPV vaccination were found to have higher levels of awareness and knowledge about HPV and HPV vaccination than those who did not consider it and were undecided (p<0.001). Those who thought it was difficult to access HPV vaccination were found to have higher scores than those who did not consider it (p<0.001) (Figure 1 and Tables 3-5).

Figure 1: Path diagram showing the model structure and standard regression coefficients.

Table 3: Factor loadings for the questionnaire, correlation between items and Cronbach alpha values when items are removed.

Items

 Factor loading Correlation between items Cronbach alpha value when item is removed
Item 1 0.493 0.409

0.814

Item 2

 0.504 0.420 0.811
Item 3 0.657 0.561

0.803

Item 4

 0.615 0.523 0.805

Item 5

 0.749 0.645

0.801
Item 6 0.812 0.714

0.797

Item 7

 0.416 0.351 0.817
Item 8 0.483 0.361

0.815

Item 9

 0.684 0.575 0.807
Item 10 0.302 0.201

0.827

Item 11

 0.305 0.203 0.823
Item 12 0.373 0.316

0.823

Item 15

 0.641 0.542 0.805
Item 16 0.594 0.500

0.806

Item 17

 0.550 0.432 0.811
Item 18 0.473 0.476

0.808

Scale Cronbach alpha value: 0.821.

Table 4: Katılımcıların sosyodemografik özelliklerine göre anketten aldıkları puanın karşılaştırılması.

 

Median (min-max)

p
Age group (year)  

0.032*
18-20

26.0 (18.0-30.0)

  
21-25

25.0 (6.0-32.0)

  
26-30

26.0 (13.0-32.0)

  
31-35

21.0 (13.0-32.0)

  
36-40

23.5 (13.0-32.0)

  
41-45

20.0 (13.0-32.0)

  
Marital status  

0.014*
Single

25.0 (6.0-32.0)

  
Married

22.0 (13.0-32.0)

  
Widow

20.0 (15.0-28.0)

  
Eğitim düzeyi  

<0.001*
Primary – secondary school

16.0 (13.0-22.0)

  
High school

18.0 (13.0-32.0)

  
College

24.0 (6.0-32.0)
Masters

28.0 (13.0-32.0)
Occupation

0.075*
Working

23.0 (13.0-32.0)
Not working

22.0 (13.0-30.0)

  
Student

25.0 (6.0-31.0)
Health worker

<0.001**
No

22.0 (6.0-32.0)

  
Yes

27.0 (13.0-32.0)
Place where lived during most of life

0.407*
City center

24.0 (6.0-32.0)
District

23.0 (12.0-31.0)

  
Village

22.5 (13.0-30.0)
Income level

0.012*
Income is less than expenses

23.0 (6.0-32.0)

  
Income is equal to expenses

23.0 (12.0-32.0)

  
Income is more than expenses

26.0 (13.0-32.0)
Has children

0.002**
No

25.0 (6.0-32.0)

  
Yes

20.5 (13.0-32.0)
Smoking

0.149**
No

24.0 (6.0-32.0)

  
Yes

23.0 (13.0-32.0)
Comorbidity

0.837**
No

24.0 (6.0-32.0)

  
Present

22.5 (13.0-32.0)
Sexual orientation

0.699**
Heterosexual

24.0 (6.0-32.0)

  
Homosexual

19,0 (18,0-29,0)

*Kruskal-Wallis test, **Mann-Whitney U test

Table 5: Comparison of the participants’ scores on the survey according to factors that may be related to them.

 

Median (min-max)

p
Given information about HPV vaccine  

<0.001*
Yes

28.0 (6.0-32.0)

  
No

21.0 (12.0-32.0)

  
HPV vaccination status  

0.320*
Yes

26.0 (6.0-32.0)

  
No

24.0 (12.0-32.0)

  
HPV vaccination status according to vaccine type  

0.336**
Not vaccined

24.0 (12.0-32.0)

  
Gardasil 4/2 doz

25.0 (6.0-32.0)

  
Gardasil 9/2 doz

29.0 (26.0-32.0)

  
Considering to be vaccined for HPV  

<0.001**
Yes

28.0 (15.0-32.0)

  
No

23.0 (6.0-32.0)

  
Undecided

23.0 (12.0-32.0)

  
Considers hard to reach HPV vaccine  

<0.001*
Yes

27.0 (13.0-32.0)

  
No

22.0 (6.0-32.0)

  

Discussion

Among sexually transmitted diseases, HPV infection is considered the most common worldwide. Persistent HPV infection is associated with more than 5% of all cancers worldwide. One of the main problems with HPV as an oncovirus is the significant difference between the time of diagnosis and the early stages of chronic infection. Early detection of HPV infection and HPV-induced lesions is critical for cancer prevention [1,3,15]. Genital HPV infection is very common among males, with an estimated prevalence of 65.2% in asymptomatic males aged 18–70 [16]. In our survey, most participants correctly answered a similar question: “HPV is a highly prevalent virus in humans.” Regarding risks to daughters, single fathers were significantly more likely to believe that their daughters were at risk for both HPV and cervical cancer. Concerns specific to single fathers included explaining the sexual nature of HPV and taking their daughters to the gynecologist for vaccination. In this respect, the results obtained for single men in our study are similar [17].

In the awareness and knowledge scale regarding HPV and HPV vaccine in this study, it was determined that those with primary school education had lower scores than those with university and master’s degrees, those with high school education had lower scores than those with university and master’s degrees, and those with university education had lower scores than those with master’s degrees. It is known that risky sexual behaviors are directly related to low education levels. Insufficient education levels negatively affect the tendency to this problem. This situation makes it difficult to prevent and treat these diseases. Similar to our findings, a cross-sectional study of 22,974 men in Denmark found that higher education was strongly associated with having heard of HPV. One study reported that men with less than a high school education were approximately 40% more likely to develop HPV infection. A cohort study of men in the United States found that having a college degree or other higher education was significantly associated with a lower risk of new infection with any type of HPV. The effectiveness of medical education has been demonstrated in an experimental study that demonstrated a statistically significant difference between pretest and posttest scores after the implementation of the education. These results support our findings. This suggests that level of education is a factor associated with HPV infection and that educated individuals have easier access to information sources and can use information more effectively. Our study found that those who were healthcare workers had higher scores than those who were not. Aslan and Bakan found that in a descriptive study conducted among health education students, including men, students who had knowledge about sexually transmitted infections had significantly higher levels of HPV knowledge. Lack of knowledge is a significant barrier to the prevention, diagnosis, and treatment of HPV infections and related diseases. The reason for the high level of knowledge among those who were knowledgeable about HPV and HPV vaccines may be attributed to the education men received as students or employees in the health field. In our study, 98% of the participants reported that they had not received HPV vaccination. In a similar study, Loke et al. reported that the HPV vaccination rate among men in the United States and Canada was very low, with rates ranging from 1.1% to 31.7% . This systematic review and meta-analysis showed that compliance with HPV vaccination was low (11% in total) among young men of working age (18-30 years). In addition, in our study, those whose income was equal to their expenses had lower scores than those whose income was greater than their expenses (0: 0.012). When the HPV and HPV vaccine awareness and knowledge level score is low, the rate of HPV vaccination is also low. From this, it is inferred that the lower the socioeconomic level, the lower the rate of HPV vaccination. The results in another study were similar. In this study, the fact that the vaccine is not covered by many health insurance companies makes it difficult for men to access the vaccine [18-24].

In this study, those who considered getting HPV vaccine were found to have higher levels of awareness and knowledge about HPV and HPV vaccine compared to those who were not considering or were undecided (p<0.001). One study found that they were more willing to get vaccinated and had higher awareness about HPV infection and malignancy [25]. Another study found that students who were more willing to get vaccinated had higher HPV knowledge scores [26]. Our findings are supported by similar studies. This situation can be explained by the fact that knowledge about the risk of exposure to HPV, especially in men, increases vaccine acceptance. In fact, when the HPV and HPV vaccine awareness and knowledge score is low, the rate of HPV vaccine application may be low. When the HPV and HPV vaccine awareness and knowledge score is high, the rate of HPV vaccine application is also high [27]. Our results were comparable to a previous study that showed that knowledge and awareness about HPV and HPV vaccine predicted it. The median score (min-max) of the men in this study group from the survey was 24.0 (6.0-32.0) and the mean (SD) was determined as 23.20. In the study group, those in the 26-30 age group were found to have higher knowledge and awareness scores than those in the 31-35 age group. In addition, men who obtained information about the HPV vaccine had higher scores than those who did not. The reason for the high mean score may be that the study was conducted in a university hospital polyclinic and most of the participants who applied were students or workers in the health field. Contrary to our findings, a study reported that 930 Singaporean men had insufficient knowledge and awareness about HPV [28]. In a descriptive study conducted by Aslan and Bakan [22] on health education students, it was found that male students had lower mean scores on the HPV knowledge level. These different results may be due to the recent increase in information about HPV and the HPV vaccine. In this study, more than half of the participants stated that HPV does not only cause diseases in women (69.8%) and that it can also cause mouth, pharynx, anus and penis cancer and genital warts in men (67.5). In a study conducted in Syria, 8.7% stated that HPV can be transmitted to both women and men [29]. According to these studies, it is thought that introducing Human papillomavirus as a cause of cervical cancer in particular may lead to the wrong perception that it is a disease specific to women only. The reason for the different results in our study is thought to be that the majority of the participants in the study are healthcare workers working at a university hospital and that there have been intensive information activities on HPV recently. It is known that HPV is a virus with more than 200 types that can be transmitted directly to the genital area, mouth and throat through sexual contact. In this study, less than half of the participants (47%) answered the question ‘HPV is transmitted only through contact with the genital area.’ correctly. According to literature data, participants stated that HPV can be transmitted sexually with a percentage ranging from 7.4% to 74.1% [30]. As can be seen, the awareness rate regarding the sexual transmission of HPV is generally low, although it varies between countries. Conservative family structures, education levels, and socioeconomic conditions can restrict society’s attitudes toward sexuality and the freedom to conduct research and discuss sexuality. In our study, more than half of the participants had a bachelor’s degree or higher, and the study was conducted in a large city, suggesting that the participants may be more aware of sexuality.

As of 2018, more than 80 countries and regions have implemented HPV vaccination programs. Although international organizations have made significant efforts to expand HPV vaccination programs, HPV vaccination rates remain below 50% in many countries worldwide. In our study, almost all participants (98%) did not receive HPV vaccination. In terms of considering HPV vaccination, the majority (78.4%) said ‘no’ or ‘undecided’. In terms of accessing HPV vaccination, more than half of the participants (56.5%) thought it was difficult. A study from sixteen European countries reported that the rate of participants considering HPV vaccination ranged from 45.6% to 79.5%. Among the Scandinavian countries, Sweden and Iceland have the highest acceptance rates of HPV vaccination. In studies, 7.7% to 37% of participants believed that the HPV vaccine could lead to early sexual orientation, while 7.9% to 68.1% stated that they avoided vaccinating themselves or their children due to safety concerns [6-8,31]. One study revealed that 62.3% of participants had heard of the HPV vaccine and 50.7% agreed to have themselves or their children vaccinated [32]. One study determined that 39.6% of participants did not get vaccinated because they did not have sufficient information and 30.8% did not get vaccinated because they thought it was unnecessary . Concerns about the potential side effects of the vaccine are an effective factor in not reaching the desired level of vaccination rates [6-9]. Research findings also reveal the need for HPV education interventions aimed at men. The level of knowledge about the HPV vaccine can affect an individual’s intention to get vaccinated. In male-specific HPV training, when the incidence of oropharyngeal and penile cancer was emphasized, it was found that men wanted to be vaccinated [33]. In a study conducted in Turkey, it was revealed that 1.1% of the participants had received the HPV vaccine. The reason for this was 94.4% of the participants stated that they were not informed, 18.7% thought it was harmful, and 4% avoided the vaccine because they were afraid of possible side effects [34]. In our study, only five (2%) of the male participants had received the HPV vaccine. In studies conducted in Turkey, the rates of receiving the HPV vaccine ranged from 1.0% to 4.3% [35]. Lack of information about HPV and the HPV vaccine prevents the desired level of interest in the vaccine both in our country and worldwide. In our country, the HPV vaccine has not yet been included in the vaccination calendar of the Ministry of Health. Although the level of knowledge about the HPV vaccine varies by country, it is generally low. Governments should make the Human Papillomavirus vaccine freely available, make it an important part of their national agenda and actively fight against it. There is generally less awareness among men that the HPV vaccine can also protect against various HPV-related cancers in men. HPV-related cancers can be largely prevented through vaccination. Vaccination protects men from developing anogenital condylomas, other malignancies related to the infection, including the penis, anus and base of the tongue. If both sexes are vaccinated, the spread of the virus decreases. Thus, it can be more effective in successfully achieving herd immunity against the virus [5-9]. In our study, most participants answered a similar question correctly. ‘HPV vaccination in men can contribute to the elimination of HPV (no cases in the community)’. HPV vaccination also helps to control the spread of HPV and reduce the overall burden of the virus on health systems. In addition, HPV-related cancer treatments are often costly and require significant resources from health systems, so vaccination against HPV may also provide cost savings in the long term. Primary and school-based HPV vaccination programs at this stage allow for higher vaccination rates and contribute to sociodemographic equality. A good solution to increase vaccination worldwide is to provide catch-up vaccination, preferably in school health systems [7-10]. The biggest problem is the lack of knowledge about preventing HPV-related pathologies. More education on this issue will increase compliance with vaccination campaigns. People who are not recommended for vaccination by their primary care physicians are less likely to receive HPV vaccination than young people who are. Vaccination of men, as well as women, is being integrated into national school-based and primary care vaccination programs worldwide, but vaccination coverage is still patchy, information is insufficient, and misconceptions persist [8-13]. Our study had some limitations. Since the data were collected cross-sectionally at a single university clinic over a specific time period, the results may not be generalizable to the entire population.

When we look at the content of our study, it is seen that healthcare workers, participants with higher income than expenses and undergraduate/graduate degrees scored higher in terms of knowledge and awareness levels about HPV and HPV vaccine. Considering the general structure of the society, male compliance with HPV vaccine is not considered sufficient. This study investigates a population of men aged 18-45, where there is evidence that compliance with vaccines is extremely poor. In order to achieve a higher level of compliance, it is important to give importance to vaccination campaigns and dissemination of information about the benefits of the vaccine as well as the risks of HPV infection.

To date, vaccination is the only way to break the chain of infection, but HPV vaccination programs, especially gender-neutral programs, are still inadequate. This makes it difficult to achieve herd immunity, especially in men who are invited to get vaccinated years after their first vaccination. There is increasing evidence that gender-neutral vaccination alone can significantly control HPV-related diseases in both women and men and can maximize the prevention of cervical cancer, especially if vaccine coverage is not high among girls in a given region. Given the current situation, it is reasonable to assume that secondary prevention will continue to be the basis for cervical cancer prevention. Another global public health goal should also be to provide scientific evidence to determine the most appropriate timing of vaccination to maximize cancer recurrence and improve outcomes of recommended treatments. Finally, health communication should also play an important role. Indeed, standardizing both the quality and quantity of information can lead to increased adherence to various vaccine awareness campaigns, which must already overcome the biases and psychological factors that complicate promotion and prevention interventions. Of course, investing in promotion campaigns, as in the case of polio, will both improve the cost-benefit balance (making this balance even more favorable than that achieved by primary prevention alone) and equalize vaccination coverage between the two sexes.

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Putting A Human Face on ‘Burnout’ in the World of Medicine: Using AI and Mind Genomics Thinking about Mind-Sets to Create a Sense of what is Going on, and What to Do

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DOI: 10.31038/ALE.2024124

Abstract

This paper explores the emerging problem of burnout among medical professionals. The approach is a combination of simulations using artificial intelligence structured by Mind Genomics thinking about mind-sets. The paper begins with using the simulations to explore the way professionals talk when in a staff meeting at a local hospital when the hospital was independent versus when it was acquired by a private equity firm. The paper continues with the exploration of mind-sets about the way medical professionals burn out, showing five different patterns or types of people. The paper continues with the use of AI to suggest messaging for the five burnout mind-sets to encourage preventive actions, and then finishes with the use of AI as a co-development coach or even partner. Through the paper the stress is on simplicity, speed, iterations to gradually improve, and the opportunity for the democratization of solving problems and advancing medicine.

Introduction

The topic of burnout and the immediate stimulus for writing this paper comes from a web posting in the news section (Table 1). Service in the medical profession at all levels and in all functions seems to be hitting the ‘respected brick wall’, of frustration. Medical professional burnout is causing higher healthcare costs, higher turnover rates, and decreased patient care. This is detrimental to the healthcare system and patients. Non-medical professionals should address mental health and well-being, empathizing with healthcare workers experiencing burnout. They should advocate for better support systems and resources for medical professionals. Concern should also arise for non-medical professionals, as a healthcare system struggling to meet patient needs due to burnout could lead to dangerous situations and subpar care [1-4].

Table 1: The Story about burnout the world of burnouts in the world of medicine.

Phase 1 – Simulations of Interviews with People

Taking a simulation approach to understanding burnout in the medical profession (Table 2) allows for a more in-depth exploration of the personal thoughts and feelings of those experiencing it. By bringing together a variety of relevant individuals, such as doctors, nurses, and administrators, and simulating discussions using advanced AI like GPT 3.5, this method delves into the underlying issues driving burnout. It personalizes the experience by allowing participants to express their true feelings, creating a more intimate and detailed understanding of the challenges they face.

Table 2: Three simulations about the causes of burnout, two from medical professionals, one from employees who are not medical professionals.

The benefits of this approach include gaining insights that may not be uncovered through traditional methods like surveys or interviews. First, simulations allow the investigator to do more deeply into inner thoughts and motivations, leading to a deeper understanding of the root causes of burnout. Second, simulation of interviews allows for a more nuanced exploration of complex relationships and dynamics within the healthcare system, providing a holistic view of the issues at hand.

When people question the validity of using simulations and AI like GPT 3.5 for understanding burnout, it can be argued that this method allows for a unique glimpse into the minds of individuals in the medical profession. By focusing on personal experiences and emotions, rather than just facts and figures, simulations provide a rich tapestry of insights that can inform future interventions and solutions. The simulated interview offers a novel way to explore the complexities of burnout and relationships within the healthcare system, paving the way for more effective strategies to address these challenges [5-8].

Phase 2 – The Value of Mind-sets and How AI Uncovers These Mind-sets Through Simulation

Carol Dweck’s seminal work on mindsets developed the concept of fixed vs growth mindsets, emphasizing the value of trusting in one’s ability to learn and evolve. This concept has been used to many fields, including medicine. Mind Genomics, as well as Moskowitz and colleagues’ research, have expanded on the concept of mindsets in healthcare, utilizing experimental design to blend multiple messages about the medical experience and analyze how people respond to them [9-11].

Researchers were able to identify fundamentally diverse attitudes among individuals by employing well established approaches such as regression analysis, grouping, and permutation. These mindsets are not necessarily innate characteristics, but rather modes of thinking that can influence behavior and decision-making.

In Phase 2 we simulate mind-sets, first using mind-sets typically discussed in the popular press because it involves the world of the everyday. We then move to simulating mind-sets focusing strictly on patterns of burnout among medical processionals (Table 3).

Table 3: Simulation of burnout mind-sets.

Simulating Solutions to Problem by the Targeted Messages Appropriate for a Mind-set

One way to begin dealing with burnout is the ‘soft’ approach of messaging. The development of Mind Genomic starting almost 40 years ago in the mid 1980’s recognized that there were different mind-sets for items as simple as toothpaste. The continuing use of Mind Genomics, and the emerge of speed, the lowering of cost, and the ease of application ended with revealing that much could be gained by working with mind-sets to craft effective messages.

Table 4 shows how generative AI can be ‘fed’ a group of mind-sets, and emerge with appropriate messages that might be used. In conventional use, these messages might either be developed with empirical-based Mind Genomics using people, or at least checked and validated later before use. Right now, however, Table 4 shows a richness of messaging to jump start the solution and can probably outperform many suggestions emerging from brainstorming. The process can be looked at as a cost-effective AI-brainstorm.

Table 4: Five AI-suggested mind-sets of doctors based on burnout patterns, and AI-suggested appropriate messaging to them to reduce burnout.

Using AI as an Invention Machine, or at least as an Invention Colleague

Generative AI, along with Mind Genomics thinking, can transform the way we address burnout by proposing new ideas that provide general guidance and explain what the innovation achieves/ The strength of generative AI in conjunction with Mind Genomics thinking stems from its potential to handle complicated human nature concerns, such as burnout, which are currently baffling and difficult to address.

Using the Mind Genomics platform such as BimLeap.com, those in the medical world may run the AI numerous times in minutes and quickly modify components of their request to adapt the suggestions to their exact needs. This ability to swiftly produce and customize burnout solutions can be extremely beneficial when made publicly available, low-cost, and user-friendly.

Table 5 shows nine innovations from one run. The objective of showing the ideas in Table 5 is to demonstrate the ease with which AI can become an integral part, even perhaps a ‘member’ in the effort to solve human-experience problems, where the issue no long is ‘factual correctness’ but rather ‘it is useful?’.

Table 5: Nine suggested innovations suggested by AI and returned to the user automatically after the ‘study’ is closed and AI has had a chance to further analyze the information it provided. The material comes from the ‘Idea Book’, and Idea Coach, attached to the project.

Discussion and Conclusions

Generative AI coupled with Mind Genomics thinking has the potential to revolutionize the way we approach burnout by suggesting innovative solutions that give general direction and outline what the innovation accomplishes. By utilizing a Mind Genomics platform like Bimieap.com, users can run the AI multiple times in minutes and easily change aspects of their request to tailor the suggestions to their specific needs. This ability to quickly generate and customize solutions for burnout can be incredibly valuable when made widely available, inexpensive, and user-friendly.

The power of generative AI in conjunction with Mind Genomics thinking lies in its ability to tackle complex human nature issues, such as burnout, that are currently perplexing and challenging to address. With the AI’s capacity to generate a multitude of suggestions and ideas, users can explore a range of innovative solutions that may not have been considered before. This opens up new possibilities for individuals to find effective ways to combat burnout and improve their overall well-being.

The democratization of expertise through generative AI and Mind Genomics platforms like Bimieap.com means that everyone can become an expert when it comes to addressing personal challenges like burnout. By empowering individuals to take control of their own solutions and decisions, this technology promotes a sense of agency and ownership over one’s well-being. This shift towards self-directed problem-solving can lead to more effective and sustainable strategies for managing burnout.

As generative AI becomes more widely accessible and user-friendly, the potential for addressing a range of human nature issues beyond burnout increases exponentially. From stress management to mental health support, the applications of AI and Mind Genomics thinking are limitless. By encouraging widespread discussion and exploration of innovative solutions, this technology has the power to catalyze positive changes in how we approach and overcome challenges in our daily lives.

Overall, the combination of generative AI and Mind Genomics thinking represents a new frontier in problem-solving and innovation, offering individuals the tools and resources they need to address complex issues like burnout in a creative and effective manner. Through this technology, we can tap into our collective expertise and wisdom, empowering everyone to become a master of their own well-being. The future looks bright with these powerful tools at our disposal, ready to shape a more resilient and thriving society.

Acknowledgments

The authors thank Vanessa M Arcenas and Isabelle Porat of the Tactical Data Group for help in preparing this and companion manuscripts in this set.

References

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  10. Moskowitz Howard R, Stephen D Rappaport, Sunaina Saharan, Sharon Wingert, Tonya Anderson, et al. (2024) “Mind-Sets for Prescription Weight Loss Products That Are Advertised Directly to Consumers: Using Mind Genomics Thinking with AI for Synthesis and Exploration.” Acta Scientific Pharmaceutical Sciences (ISSN: 2581-5423)
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Putting a Human Face on Legal Disputes: Eviction for Non-Payment of Rent Simulated by AI and Mind Genomics Thinking

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DOI: 10.31038/ALE.2024123

Abstract

Using generative AI (Chat GPT3.5), the paper shows how a legal case such as eviction for non-payment of rent can be ‘brought to life’. The first simulation shows how AI can be used as a preparatory device for the Mind Genomics experiment, thus making the lawyer’s job easier when using Mind Genomics for a legal case. This first simulation focuses on AI-created questions and answers, and the ‘guesstimate’ by AI as to the likelihood that a jury will find ‘for the plaintiff’ vs ‘for the defendant’. The second simulation focuses on different reasons for the eviction, and AI-based analyses of legal aspects vs ethical aspects. This second stimulation demonstrates the ability of AI to consider the nuances of a case. In both simulations the focus is on types of issues that are best described by phrase ‘subjective, and open to interpretation.’

Introduction

As legal education evolves, law schools are placing greater emphasis on equipping students with the skills to critically analyze intricate legal cases. Traditional methods for teaching law methods frequently rely on rote memorization of statutes and procedures. However, a growing number of educators are now embracing experiential learning opportunities, allowing students to hone their analytical skills in practical contexts. In this new era of legal education, innovative technologies such as generative AI are increasingly being utilized to simulate legal cases, enabling students to navigate real-world scenarios effectively. When combined with the emerging science of Mind Genomics the new mix enables students, or indeed anyone, to gain a human perspective on the law, viz., put a human face on the facts through simulation. The simulation allows learning by asking ‘what-if’ questions, not about the case law itself, but about the softer, human aspects [1-5].

This paper shows two simulations of the same case, where a landlord seeks to evict tenants who have fallen behind on rent for two months due to job loss. The case itself is rich with legal versus emotional and moral complexities. In the classroom, students might end up relishing the back-and-forth analysis, as they engage in a spirited discussion, exploring the pertinent legal principles, the rights of both landlords and tenants, and possible solutions to the issue at hand. This exchange of ideas enables students to examine various viewpoints, sharpen their arguments, and gain a richer insight into the intricacies of legal decision-making.Can we make this back and forth into an AI application, using currently available LLM’s, large language models? As this paper shows, we can do so in ways which are staggeringly fast, inexpensive, scalable, and adaptable to many situations [6-8].

Simulation in the legal field breathes life into cases, offering a vivid experience which textbooks and lectures often fail to convey. Generative AI lets law students explore the intricate complexities and subtleties of real-world legal dilemmas through simulated cases. In this scenario, the AI offers clarity on landlord-tenant laws, the eviction process, and the delicate balance of interests between property owners and tenants.Students can delve into various viewpoints and possible results of the case through simulation. The legal rights and responsibilities of both landlords and tenants can be examined, alongside an evaluation of the ethical implications stemming from the decisions made by each party. This practical method sharpens critical thinking and fosters a richer comprehension of legal principles at work.

This paper explores the two simulations, based on the same ‘facts of the case’. Simulation 1 deals with what to present to a jury to get the jury to vote for the client. Simulation 2 moves more deeply into the role of the lawyer as an advocate for their client.

Simulation 1: Mr. Owner vs Mr. & Mrs. Tenant – Rent Dispute and the Search for Discovering Powerful Messages Resonating with the Jury

We begin with the facts in the case, shown in Table 1. The stimulation is set up to provide messages that can be tested by the emerging science of Mind Genomics, to identify those specific messages which will resonate with a jury [9-12].

Table 1: Facts of the case and instructions to AI (Simulation 1)

The actual Mind Genomics experiment with real people (not shown) follows a straightforward process. The steps are as follows:

  1. Select a topic
  2. Develop four different questions about that topic which give a human face to the topic
  3. For each question develop four different answers to the question, phrased as stand-alone phrases which paint a word picture
  4. Combine the 16 elements into small, easy-to-read vignettes comprising a minimum of two and a maximum of four elements. The composition of the 24 vignettes to be evaluated by a single respondent follows an underlying experimental design. Each question contributes only one element to the vignette. Across the set of 24 vignettes each of the 16 elements appears 5x and is absent 19x. Mathematically the structure of each respondent’s set of 24 vignettes is identical, but the permutation ensures that the vignettes are different across all respondents.
  5. Each respondent evaluates a permuted set of 24 vignettes, using a simple rating scale. The vignette is a stand-alone composition, easy to read. The respondent requires about 3-4 seconds to scan the combination of messages and assign a rating.
  6. The data are put into a simple data base to prepare them for statistical analysis. Ordinary least-squares, dummy variable regression analysis shows the driving power of each element, first for each respondent, and then for groups of respondents defined by methods such as cluster analysis.

Up to now the creation of raw material, questions especially, has emerged as a stumbling block. For whatever reason, people are intimidated. Over a 24-year period, 1998 to 2022, the same plaint was heard, always by the researcher using the tool. It was simply daunting, although children had fewer problems than did adults, and especially far fewer problems than did professionals. Professionals wanted perfection in the vignette, ending up enamored with overly polished, wordy paragraphs, hard to read, hard to test more than a few.

The introduction of AI in 2022 revolutionized this emerging science of Mind Genomics. As Table 1 showed it is straightforward to come up with the statement of the ‘facts of the case’. It is the creation of the questions and elements (answer) which are difficult.

Table 2 shows what emerged after one iteration requiring 10-15 seconds. These four answers are statements about the case, and the AI’s ‘guesstimate’ about the jury’s decision based on each answer. The key benefit to critical thinking is the ability of AI to jump-start the thought process. To ‘have fun’ one could iterate, with each iteration requiring the 15 seconds. An encyclopedia of questions and elements (answers) would emerge in about 10 minutes or shorter. The enterprising user could change the facts in the case as well and explore different ‘what if’ aspects, using the Idea Coach features.

Table 2: Simulation of four questions and four elements (answers to each question), prepared for a Mind Genomics experiment with actual people (Simulation 1).

The effort is finished, whether one iteration or 100 iterations, the results of each iteration are stored on an Excel spreadsheet, with one tab dedicated to each iteration. Thus, it is possible to run many iterations, acquire the data, but leave the subsequent analysis to an off-line effort, that effort taking about 5-6 hours. The results are returned to the user by email. The remainder of this simulation is generated after the study preparation is closed. AI then goes over its own questions and answers and provides a summarization and deeper analysis shown in Table 3, and beyond. Table 3 presents the first set of information,

  1. Key Ideas: AI can provide valuable insights into key ideas by analyzing large amounts of data and identifying patterns and trends. It can summarize complex concepts and present them in a clear and concise manner. This can help individuals better understand key concepts and make informed decisions.
  2. Themes: AI can also identify common themes across different sources of information, allowing for a deeper understanding of the subject matter. This can help in identifying trends and patterns that may not be apparent at first glance.
  3. Perspectives: AI can provide different perspectives on a topic by analyzing a wide range of sources and presenting diverse viewpoints. This can help individuals gain a more comprehensive understanding of the subject matter and consider different angles.
  4. What is missing: AI can also highlight what is missing in a particular discussion or research study by pointing out gaps in information or potential areas for further exploration. This can guide researchers in identifying areas that need further investigation.
  5. Alternative viewpoints: Additionally, AI can present alternative viewpoints on a topic by analyzing conflicting information and presenting different sides of the argument. This can help individuals consider different perspectives and make well-informed decisions.

Table 3: AI summarization of simulation 1 in terms of key ideas, themes, perspectives, what is missing and alternative viewpoints.

Table 4 compares interested audiences versus opposing audiences regarding the material presented in Table 3. AI can expand the topic by questions and answers tailored to specific interested audiences, allowing for a more personalized and engaging experience for learners. AI can create content that is relevant and meaningful content. increasing their motivation and interest in the material. Going a step further, AI can also identify prospective opposing audiences by presenting diverse perspectives and challenging viewpoints. By generating questions and answers that provoke critical thinking and debate, AI can stimulate discussions and encourage individuals to consider alternative viewpoints. This can lead to a more well-rounded understanding of the topic and foster a culture of open-mindedness and tolerance.

Table 4: AI synthesis of interested versus opposing audiences, based on material in Table 3.

The final analysis as of this writing (November 2024) is the suggestion of innovations. The initial suggestion of innovations produced a list of AI-generated innovations. During the summer of 2024, the Mind Genomics system was modified so that the post-creation of the study (not-yet-run with people) would offer a far more complete analysis of each innovation that was being created and offered to the user. These nine aspects appear below and are shown in Table 5 for each of the four innovations.

  1. Specific suggestion: AI can provide specific and targeted suggestions based on data analysis and patterns
  2. Explanation why the suggestion is relevant:
  3. Importance and uniqueness of the suggestion:
  4. Social Good resulting from the suggestion.
  5. Slogan which emblemizes the suggestion:
  6. Investment pitch:
  7. Potential investor pushback:
  8. Answer to the potential investor pushback:
  9. Compromise, go forward positioning:

Table 5: The four innovations suggested by AI and an AI ‘work-up’ of each innovation

Simulation 2 – Dealing with the Same Issue, but From the Point of View as the Lawyers Helping Both Sides as ‘Trusted Parties’

In this simulation we move backwards, away from the situation of a lawyer trying to anticipate what to say to the jury (Mind Genomics approach), to understanding the ‘facts in the case’ from the point of view of the client. Here the lawyer becomes a trusted friend. The process becomes more personal and empathic.

Table 6 shows the ‘facts of the case’ and the instructions to AI to generate the relevant questions and answers. Table 7 shows nine different questions and the structured answers to those questions.

Table 6: Facts of the case and instructions to AI (Simulation 2)

It is worth noting here that generative AI such as Chat GPT3.5 used here, can be instructed to simulate different people talking to each other, e.g., listening in as a ‘fly on the wall’ to the lawyer’s conference.

Table 7: Nine relevant questions generated by AI regarding the case, with answers simulated by AI with respect to from the legal standpoint and the moral standpoint.

Discussion and Conclusion

Simulations are crucial for students to understand the intricacies of legal decision-making, enabling active engagement with complex scenarios and practicing their knowledge in real-world contexts. These simulations enhance students’ understanding of the law and its real-world uses, as well as their critical thinking and problem-solving abilities. The future of law schools will likely employ diverse techniques and technologies, guiding students to think critically about complex issues. Mind Genomics, enhanced by AI, presents a groundbreaking solution for the legal system, offering students an engaging and interactive approach to grasp the intricacies of legal cases. This technology has the potential to reshape the legal landscape, fostering quicker case resolutions, enhancing transparency, and expanding access to justice for everyone. Mind Genomics and generative AI can craft realistic legal scenarios by examining the many factors which shape legal decision-making, including case law, evidence, and ethical considerations. These scenarios can provide students with practical experience to enhance their legal reasoning skills in a simulated environment.

Mind Genomics, powered by AI, has the potential to revolutionize the legal profession by improving decision-making, streamlining case resolutions, and ensuring justice is served efficiently and transparently. This technology would benefit students by providing interactive learning tools, real-world case studies, and hands-on experience, while professors would have access to cutting-edge research tools and real-time data. The implementation of Mind Genomics could transform the legal practice, leading to more efficient case resolutions, improved transparency, and greater access to justice for all individuals.

Reflection and debriefing sessions on simulated legal cases can help students improve their legal reasoning abilities, bridge the gap between theoretical legal knowledge and practical application in real-world scenarios, and develop a deeper understanding of human factors involved in legal decision-making. At the same, it might well be productive is during the application of AI to legal issues the user requests both legal and ethical/moral considerations as separable steps in the process.In conclusion, using technology-driven simulations in legal education can promote active learning, enhance critical thinking skills, and provide practical experience in legal decision-making, equipping students for successful careers in law.

Acknowledgments

Author Moskowitz wishes to acknowledge the help of the audience at his lecture at the law school in of the University of Kragujevac, November, 2024. Many of the points of view regarding ethics and morality and the law were challenged by the students and professors, leading to refinements in the authors’ thinking.

The authors would like to thank Vanessa M. Arcenas and Isabelle Porat for help in preparing this manuscript for publication.

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Designing Eco-Serbia for High School Students: Using AI Simulation with Mind Genomics Thinking and Technology to Inspire Critical Thinking by a Gamifying a Topic

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DOI: 10.31038/MGSPE.2024443

Abstract

AI (Chat GPT3.5) embedded in the Mind Genomics platform (Idea Coach feature of BimiLeap.com), was used to synthesize a teaching approach for critical thinking. The exercise, strictly based on interaction with AI, was guided by the request of students in the Gymnasium of Novi Sad, Serbia, to create a tool which would allow them to experience critical thinking in a gamified manner. The process and prompts are presented in this paper, with the path to create a new game, Eco-Serbia. The paper shows how to introduce the topic, the power of AI to simulate discussions about a topic, to create the specifics for the game, and then shows additional analyses. These additional analyses, done after the project has been ‘closed’ by the user. include showing how to discover key ideas, themes, perspectives, and types of responses by different audiences. The paper finishes with a detailed opportunity analysis of four innovations about eco-Serbi as suggested by the AI [1-4].

Introduction

Teaching gymnasium-level students critical thinking is vital; it empowers them to analyze information, evaluate arguments, and reach informed conclusions. Developing critical thinking skills enables students to sharpen their awareness of the world around them, prompting them to question assumptions, form their own opinions, and approach challenges with innovative solutions. The result is enhanced academic achievements while being equipped with the skills to navigate complex challenges in a diverse and interconnected world [5-9].

Integrating critical thinking into the high school gymnasium curriculum ignites student creativity, inspiring them to generate innovative ideas and solutions. Through the application of critical thinking skills across subjects such as science, literature, and history, students increase their understanding, interact meaningfully with the material, and enjoy a sense of curiosity and intellectual exploration. This method boosts academic success while igniting a lasting love for learning, inspiring students to engage in critical thinking across all areas of their lives.

Gamifying Strategy: Create a Game About the Topic, One Requiring and Rewarding Critical Thinking

Gamifying by using AI to create a game for eco-tourism in Serbia adds a level of excitement and engagement for the students. By turning the process into a game, it becomes more interactive and enjoyable, making the learning experience more fun and memorable. The outcome of making the AI effort focused on gamifying the project is more motivated students, enthusiastic about the task at hand. They will be more likely to invest their time and creativity into the project, leading to a higher quality final product [10-13].

When our Serbian gymnasium students in Novi Sad use AI and Mind Genomics-based thinking to create a game for eco-tourism in Serbia, we expect them to feel a sense of accomplishment and pride in their work. They will likely be more excited and engaged in the project, as they see it as a fun challenge rather than a tedious academic task. By gamifying the effort, they will be more likely to collaborate with their peers, think outside the box, and push themselves to create something unique and innovative [14-17].

By using AI to transform the creation of the eco-tourism game for Serbia into a gamification project, we hope to foster creativity, critical thinking, and a passion for innovation among our students. This approach will not only make the learning process more enjoyable and engaging but also equip them with valuable skills and experiences to apply in their future endeavors.

The remainder of this paper shows the approach, done virtually all with the aid of generative AI, specific Chat GPT3.5, using the Mind Genomics platform (www.BimiLeap.com). The AI is available through the Idea Coach feature. The set-up portions of the program are done by prompts easily created by the user. The detailed analysis after the program is closed, is done by a set of embedded prompts in the BimiLeap. Program and returns automatically for each iteration after the project is closed. The results done here required about 90 minutes. The deeper analysis was returned after the AI had finished its deeper analysis.

Phase 1 – ‘Listening in on the Conversations Among 10 Students When They Hear About the Project

Observing a conversation from the sidelines offers a glimpse into the thoughts, emotions, and viewpoints of individuals regarding a specific subject. The genuine voices of individuals discussing a topic offer an authenticity that written sources often lack. The subtleties of tone, body language, and emotions in spoken conversations enrich the understanding of the topic at hand.

Engaging in conversations about a topic fosters a deeper connection and understanding that reading from a textbook or article simply cannot achieve. Hearing firsthand accounts and personal anecdotes allows us to connect more deeply with the topic, revealing its significance in our own lives. Conversation’s human element weaves empathy and understanding into the fabric of discourse, essential for unraveling the complexities of any topic.

Additionally, being a ‘fly on the wall’ enables us to observe the intricate dynamics of group communication and the interplay of diverse perspectives and opinions. This opportunity allows us to broaden our perspectives and question our beliefs as we engage with the diverse viewpoints shared in the discussion. Engaging deeply in the conversation allows us to grasp a fuller and richer understanding of the subject matter.

In the realm of gamification, listening to discussions on a topic can offer essential insights for crafting captivating and interactive experiences. By grasping the subtleties of how individuals discuss and interact with a subject, game developers can craft gameplay that is more immersive and meaningful. Incorporating real-life dialogue and scenarios into games can make them more authentic, relatable, and ultimately more enjoyable for players. Table 1 shows the instructions for the AI to be a fly on the wal, and the results generated by AI.

Table 1: The fly on the wall’ strategy to simulate a conversation about Eco-Serbia.

Phase 2 – Using Detailed Instructions to AI to Create the Game

To craft a game, the AI must produce questions that are both stimulating and demanding, while also being enjoyable and captivating for the players. Incorporating elements of surprise, humor, or suspense into the questions can achieve this, along with varying the difficulty level or topic to engage a diverse group of players. The goal is to craft an engaging and lively game experience that captivates players and encourages them to return for more excitement. Using the AI’s cognitive skills and computational strength, game developers can craft tailored and immersive gaming experiences that resonate with the varied preferences and interests of players. The aim is to use AI’s potential to enrich the gaming experience, crafting more interactive and enjoyable games into which players can immerse themselves (Table 2).

Table 2: The AI-simulated created of the Eco-Serbia project, showing the instruction to the AI, and the steps returned to create the game.

Phase 3 – ‘Harvesting and Answering Questions Generated by AI at the End of Iteration 1, as well as Questions Asked in Iteration 2, and Finally Questions Emerging When AI Reviewed the Material After the Study was Closed

AI in platforms like Idea Coach on BimiLeap.com consistently prompts relevant questions and when instructed answers to those questions. These questions highlight important issues related to the topic. Inserting these questions into a BimiLeap iteration is simple, regardless of the topic’s origin or relevance. The user directs the AI, through the Idea Coach feature, to respond to the question using Chat GPT3.5 in a format relevant to the user. Table 3 shows 25 questions and answers generated by AI and answered by AI. These 25 questions and answers may overlap, but in the interests of showing what can be learned, all questions and answers are included.

Table 3: AI generated questions about the topic, and AI generated answers to those questions.

Phase 3: AI as Teacher of Critical Thinking by Reviewing the Original AI Output Automatically After the Study is Closed for Further Iteration

What are the Key Ideas?

The key ideas generated by AI in a topic provide a concise summary of the main concepts and themes within the subject matter (Table 4). Knowing the key ideas allows students to quickly grasp the most important information, enabling them to better understand and absorb the material. To find these key ideas, students can use tools such as text analysis software or utilize critical thinking skills to identify recurring themes and main points. Once the key ideas are determined, the next steps involve further exploring and analyzing them to deepen comprehension and stimulate critical thinking.

Table 4: Key Ideas

Finding key ideas and utilizing them to teach critical thinking is essential for developing students’ analytical skills and ability to extract meaningful information from complex topics. By focusing on key ideas, students learn to identify the most important elements of a subject and distinguish between relevant and irrelevant information. This process encourages critical thinking by prompting students to evaluate, question, and form their own opinions based on the key concepts presented.

What are the Themes and How do These Themes Manifest Themselves as Perspectives?

By exploring the themes highlighted by AI in the Mind Genomics analysis, individuals uncover a richer understanding of the patterns and foundational concepts embedded in the data. Examining the expression and interconnection of these themes allows researchers to glean valuable insights and pinpoint key takeaways from the study. A young researcher in Serbia can gain fresh insights and inspiration by exploring the list of themes, enriching their grasp of the subject matter. Through the examination of themes, students enhance their critical thinking abilities, recognizing patterns, forging connections, and deriving conclusions from the presented data. This approach prompts students to engage in analytical thinking, evaluate information with a critical eye, and present well-reasoned arguments to back their conclusions. The list of themes can act as a springboard for further research, igniting curiosity and encouraging students to explore the subject matter more deeply.

Students can enhance their critical thinking skills by examining the underlying assumptions and implications of each theme in the list. They can investigate the links between various themes and reflect on how these elements enhance the overall comprehension of the subject. Students can assess the relevance of each theme, gauge its impact on the research findings, and pinpoint any gaps or inconsistencies that may arise.

The themes identified through AI analysis provide a useful resource for enhancing critical thinking skills in education. Through a thoughtful exploration of themes, students can sharpen their analytical skills, foster logical thinking, and empower themselves to make well-informed decisions. This approach deepens understanding of the subject while inspiring students to tackle research and problem-solving with critical and analytical thinking (Table 5).

Table 5: Key themes recurring in the material generated by AI about the Eco-Serbia project, and the emerging perspectives.

Interested Audiences versus are Opposing Audiences

Understanding the interested and opposing audiences for the Eco-Serbia project, along with its gamification element, can significantly enhance our efforts in multiple ways. Identifying the audience interested in the topic enables us to customize our messaging and strategies, ensuring we engage and educate them effectively. This understanding enables us to craft focused campaigns and initiatives that connect with our audience, fostering increased participation and support for the project. Recognizing opposing audiences allows us to foresee challenges and objections, empowering us to tackle them head-on and refine our strategy to reduce resistance.

Additionally, grasping the perspectives of both supportive and opposing audiences can enhance critical thinking skills for participants and stakeholders alike. Through the examination of various viewpoints and the recognition of possible conflicts, individuals are prompted to engage in critical thinking regarding the project and its consequences. This cultivates a deeper insight into the relevant issues and promotes engaging conversation and discussion. Engaging with diverse viewpoints and opposing arguments allows participants to sharpen their analytical skills and make informed decisions regarding the project.

Integrating AI to analyze interested and opposing audiences in the Eco-Serbia project can significantly boost the effectiveness of its gamification elements. By grasping the preferences and viewpoints of various audience segments, we can customize the gamification elements to resonate more effectively with their interests and motivations. This focused method increases engagement and participation, paving the way for a more effective gamification strategy. By examining opposing viewpoints, we uncover potential challenges or concerns in the gamification process and tackle them proactively, creating a smoother and more enjoyable experience for all participants

Overall, it is important to address these potential oppositions by promoting the benefits of critical thinking, problem-solving, and environmental awareness, and by emphasizing the positive impact that the game can have on students’ education and understanding of the world around them (Table 6).

Table 6: Interested versus opposing audiences

Suggested Innovations – Analysis of Each from the Viewpoints and Business Opportunity, Respectively

The AI analysis prompts individuals to critically evaluate the project’s business dimensions. These dimensions include social aspects, uniqueness as well as business-case aspects. AI encourages individuals to foresee and tackle possible criticisms from investors, competitors, and other essential stakeholders, enabling researchers to craft a stronger and more convincing case for the gamification of the Eco-Serbia project. This thorough examination not only guides decision-making but also sharpens individuals’ capacity to strategically maneuver through intricate and layered challenges, ultimately boosting their critical thinking abilities along the way.

AI encourages individuals to question assumptions, challenge prevailing beliefs, explore alternative perspectives, and engage in strategic decision-making. The result is a culture of innovation, collaboration, and sustainability, crucial for tackling complex environmental challenges. Through critical analysis and reflection, individuals can cultivate stronger, more effective, and sustainable solutions for the Eco-Serbia project and beyond (Table 7).

Table 7: Deep AI analysis of four AI-suggested innovations.

Discussion and Conclusions

Making intellectual topics into games has greatly improved critical thinking by letting people interact with and think about knowledge in a fun and engaging way. Adding game features like challenges, puzzles, and competition encourages people to think critically and solve problems in order to win the game. This process helps people become better at analyzing things and pushes them to look at complicated issues from different points of view and think differently.

Gamification lets people use their critical thinking skills in the real world, making it easier to learn about and understand complicated topics through hands-on activities. People can improve their critical thinking skills in a safe and controlled setting by putting decision-making, strategy-building, and information processing into games. This makes them better at critical thought and gets them ready to use these skills in school, work, and personal life.

Using gamification as a tool helps people think more critically and interact with and learn more about intellectual topics in a more interesting and engaging way. This method turns learning into something fun and satisfying, which helps people understand and appreciate difficult topics more. By turning intellectual topics into games, people can connect with information in a fun and active way. This encourages exploration, analysis, and questioning, which leads to a deeper and more complete learning experience.

Adding game elements to educational topics could change the way people learn and improve their ability to think critically. Adding game features to intellectual and educational settings makes it easier for people to interact with and learn more about difficult topics in a fun and active way. This not only improves their critical thinking, but it also gives them the tools they need to deal with problems, face difficulties, and think deeply about different situations. By turning intellectual topics into games, people can start an interesting and exciting learning journey that can change them.

Acknowledgment

The authors gratefully acknowledge the ongoing help of Vanessa M Arcenas in the preparation of this manuscripts of the others in this grouping.

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Simulating and Stimulating Critical Thinking Using Mind Genomics Turbocharged with AI: Homelessness in the America of Tomorrow, 2030

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DOI: 10.31038/MGSPE.2024442

Abstract

This paper presents a new, AI-based computer approach to simulate as well as stimulate critical thinking and generate potential innovations in a topic chosen by the researcher. The presentation here speculates with the help of AI regarding the outlook for homelessness in th United States. The paper shows the different stages of critical thinking for this topic and provides examples of how to develop a critical thinking mind-set. The technology is resident in a publicly available website, www.BimiLeap.com.

Introduction

Homelessness is a widespread issue in the United States, with approximately 500,000 people experiencing homelessness on any given night. The causes of homelessness are complex and varied, including factors such as lack of affordable housing, poverty, mental illness, and substance abuse. Those experiencing homelessness often face numerous challenges, including lack of access to necessities such as food and shelter, as well as barriers to employment and healthcare [1-4]

Artificial intelligence (AI) has the potential to play a significant role in addressing homelessness in the future. AI can be used to analyze data and identify trends in homelessness, allowing policymakers to make more informed decisions about resource allocation and intervention strategies. AI can also be used to develop predictive models that can anticipate homelessness trends and help target interventions to those most at risk. Additionally, AI can be used to streamline processes and improve efficiency in the delivery of services to individuals experiencing homelessness [5-9]

This paper presents an attempt use generative AI (Chat GPT3.5) accessed and prompted by Idea Coach, a feature in the Mind Genomics platform, BimiLeap.com. the approach shows how to use AI to study homelessness as a problem, using simulations (fly on the wall technique), AI-generated questions and answers, and then critical thinking about the results suggested by AI. The paper finishes with using AI to generate 10 suggested innovations, and for each innovation the paper shows how AI can dissect the innovation into its components and business opportunities.

Phase 1 – The Fly on the Wall Strategy Simulated through AI

The phrase “fly on the wall” refers to being able to listen in on a talk without being seen. In this case, “fly on the wall” refers to both that action and knowing what someone is thinking and why they are saying something. Being a fly on the wall in a meeting gives one a unique view of conversations and exchanges that might not have been seen otherwise. Oen can learn more about people’s goals and motivations by listening in on their conversations and hearing their private thoughts. This can be especially helpful when talking about touchy or controversial issues, because it lets one hear other points of view without getting involved in the conversation directly (Table 1).

Table 1: ‘Fly on the wall’ strategy at local meeting in ‘Smallville, USA, a town with coping with homelessness.

Besides that, being a fly on the wall gives one some privacy and objectivity. One can listen to the discussion without taking part and form their own opinions based on what they hear. This can help one learn more about a complicated issue, like homelessness in oner neighborhood, by gathering information about it. Being a “fly on the wall” can also help one figure out political goals and hidden agendas that are not talked about publicly. One can find out about underlying tensions or alliances which may affect decision-making by listening in on private thoughts and responses. This is often very important for getting a sense of how a meeting or group really works. Being a “fly on the wall” also lets one get a better sense of how people talk to each other and who has power in a group. One can figure out alliances and hierarchies that aren’t clear at first glance by watching who asks what questions and how others answer. This can help one understand how choices are made and who has power in a certain situation [10-13] (Table 1).

Phase 2 – Jumpstarting Learning by Instructing the AI to Create Sets of 15 Questions and Answers

Working with AI to create questions and answers benefits critical thinking by promoting creativity, prompting exploration of different angles, improving problem-solving abilities, enhancing analytical skills, and fostering innovation. The Idea Coach feature of BimiLeap.com, the Mind genomics platform, can come up with 15 different questions detailed answers in less than 30 seconds. For a deep analysis in a very short time, the process can be done almost effortlessly, with an iteration completed every half minute, for a total of 300 questions and answers in 10 minutes. Across all 300 questions about a third to a half will be unique, and not repeats. The benefit for critical thinking is clear, if only that AI can, in a virtually automated fashion, produce thousands of questions and answers in an hour, sufficient for a rapid education in the topic [14-17] (Table 2).

Table 2: Questions and Answers from Iteration #21.

Critical Thinking – Letting AI Review Its Own Questions, and Identify Questions Which Were Missing

After the ‘study’ is closed, the AI reviews the material it created, shown in Tables 1 and 2. AI then identifies questions that may have been ‘missed’ being asked, and presents them. Table 3 shows the remaining 10 questions identified by AI in its ‘self-review’. These 10 questions were run separately, with the same instructions as given in Table 2. Table 3 shows the remaining 10 questions and their answers. The number is consistent with that used in Tables 1 and 2 [18-21].

Table 3: Questions discovered by AI not to have been asked previously by AI, with their AI-generated answers.

Critical Thinking – Key Ideas and Themes in the 15 Original Questions and Answers

Dividing the topic of homelessness into key ideas, identifying themes, can significantly enhance critical thinking skills, particularly when working on a do-it-yourself project. By delving into various perspectives, one can gain a deeper understanding of the complexities surrounding the issue of homelessness and develop a more well-rounded approach to addressing it. This process forces individuals to think critically about the root causes of homelessness, the societal factors at play, and the potential solutions that can be implemented. Table 4 shows the key ideas and the themes [22-24].

Table 4: AI-abstracted key ideas and themes.

Critical Thinking – Alternative Views and Systematizing Them Through Perspectives

People are pushed to question their own views and biases when they look at things from different points of view. This leads to a more open-minded and objective analysis of the issue at hand. By doing this, people not only improve their minds, but they also start to think about more options and answers. Finding perspectives in the topic of homelessness can also help people organize their thoughts and ideas in a way that makes sense. This makes it easier to see patterns and links between different points of view (Table 5).

Table 5: Alternative viewpoints regarding topics involved in homelessness, and formalized analysis of these differences through perspectives.

By looking at things from different points of view, people can see the problem of homelessness from more than one angle, which helps them understand and care about those who are homeless. People can think about different points of view and approaches, which can help them come up with more creative and useful answers. Overall, breaking up ideas into different points of view, formalizing the through perspectives and exploring themes forces people to think more deeply and critically [25-27].

Critical Thinking – Which Audiences are Likely to be Interested Versus Which Audiences are Likely to Oppose

Understanding the accepting and opposing audiences allows one to more deeply understand multiple perspectives before making decisions related to homelessness in Smallville township. By understanding the potential supporters and detractors of each proposed solution, one can anticipate challenges and resistance, and adjust the approach accordingly. This level of critical thinking helps identify potential pitfalls and ensures that the proposed solutions have a higher chance of successful implementation (Table 6).

Table 6: Responses of interested versus opposing audiences.

The understanding of responses by different audiences lets one consider the feasibility and impact of each solution in a more nuanced way. By recognizing the diverse viewpoints within the community, one ca approach problem-solving with a more holistic understanding of the situation. This process ultimately leads to more effective and sustainable solutions for addressing homelessness in the Smallville township (Table 6).

Critical Thinking – ‘Deep Dive’ Analysis of 10 AI-suggested Innovations

With AI’s ability to analyze vast amounts of data and identify patterns, it can suggest innovative solutions that may have been overlooked by human researchers. Additionally, AI can provide real-time feedback on the effectiveness of these inventions, allowing for quick adjustments and improvements. This can greatly accelerate the progress in tackling homelessness and provide more efficient and effective solutions for those in need.

However, it is important to critically analyze the potential drawbacks of relying too heavily on AI in addressing homelessness. Whereas AI can provide valuable insights and recommendations, it may lack the empathy and understanding that human intervention can offer. It is essential to strike a balance between using AI as a tool for innovation and ensuring that human intervention and support are still prioritized in addressing the complex and nuanced issue of homelessness.

A standard feature of the BimiLeap.com platform is that at the end of each iteration, and after having reviewed all the information generated in that iteration, the AI suggests innovations, and for each innovation does a ‘deep dive’. That ‘deep dive’ looks at the nature of the innovation, the explanation of the innovation, its importance, uniqueness, attractiveness and degree of expected social good. The ‘deep dive’ finishes with slogans, and then with the different facets of a ‘business pitch’. Table 7 shows the real depth of the analysis for the 10 innovations [28-33].

Table 7: ‘Deep dive’ analysis of 10 innovations generated during one iteration (#21).

Discussion and Conclusions

Critical thinking is essential for solving complex issues like homelessness, as it helps individuals weigh options based on facts and common sense. It helps avoid relying on assumptions or stereotypes, and allows individuals to see things from different perspectives. Critical thinking also helps individuals question the status quo and come up with new solutions, ensuring solutions are based on diverse experiences and perspectives. It also encourages a mindset of continuous learning and improvement, allowing for flexible and adaptable solutions. For instance, if one is unfamiliar with homelessness, critical thinking can help them understand the main causes, current laws, and available tools to help homeless individuals find stable housing. Overall, critical thinking is a useful skill for people who want to deal with tough social problems like homelessness. People can make real changes to help end homelessness in their community by keeping an open mind, asking deep questions, looking for solid proof, and coming up with creative solutions.

Within this framework, the strategy of simulating and stimulating critical thinking using the Mind Genomics platform provides a promising tool, perhaps a tutorial. In a matter of minutes up front, and with output later on, the user can try out one, two, even a dozen or more alternative scenarios and issues, with the analysis automatic, simple, rapid, easy to understand, and occasionally even profound.

Acknowledgments

The ‘research stimulations’ for this paper emerged from iteration 21 (Results 21_ using AI access through the Idea Coach feature of BimiLeap.com. The AI, Chat GPT3.5, provided all the AI-based material. Bimileap.com is openly available for public use at a modest platform fee. BimiLeap.com is the platform for the emerging science of Mind Genomics, the inspiration for the work shown here.

The authors wish to thank Vanessa M. Arcenas for her ongoing help in preparing this and other manuscripts in this series.

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Subsurface Geopressure Prediction: Perception and Pitfalls

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DOI: 10.31038/GEMS.2024674

Abstract

In this short article, a review of the subsurface geopressure forecast’s characteristic in relation to geological building blocks is briefly presented. Geopressure is the driving mechanisms of generating and migrating of oil and gas to their traps. Geopressure is a double edge sword. Most of the large hydrocarbon pools are embedded in this pressure zone, and on the other hand it can be a serious hazard for drilling crews and properties. The lack of understanding the prediction process can resulted in unintended miscalculation and ambiguous interpretation. The science of predicting the sedimentary column pressure profile drift is widely diverse and mostly driven by algorithms rather than the exploration prospect’s geological settings.

Introduction

Pore pressure prediction (PPP) is a process synonym of estimating the subsurface pressure before and post drilling. However, predictions should apply only if the calculation takes place before drilling. On the other hand, any estimation or assessment of the pressure from drilling records and logs data should not be considered as forecasts. They are pressure calibration or simulation instead of prediction. Subsurface pressure compartments are the response of sedimentation process, that lead to compaction and burial. Sand and shale/clay dominate most of the deltaic and shallow marine environments. Compaction due to sediment load leads to fluids expulsion out of the rock matrix that leads to increase pressure. Terzaghi and Peck (1948) [1] illustrated this process with a mechanical device filled with water. Most of the geopressure scholars believe the clastic sedimentary column is divided to two sections: Normal and Abnormal pressure (geopressure) zones. Eaton (1975) [2] made a successful leap to build the base model of calculating subsurface pressure increasing with depth using petrophysical properties (sonic, resistivity and density). The assumption of two main subsurface zones were utilized in his assessment as well. Shaker (2015) [3,4] recognized a new concept in the presence of 4 pressure zones in offshore and 3 zones in onshore in the clastic sedimentary columns. Based on the new established subsurface compartmentalization, a geological based prediction model of the subsurface geopressure is attained. This was an eye opener for some of the unintended pitfalls of geopressure prediction methods.

The Basic Geological Framework

Sediments reach the depositional basin carried by water through the water shed feeder areas. Suspended detrital grains begin the slow consolidation and compaction throughout time and additional load. During the periods of high sea stand, capable competent seals form. The top seal represents the choking barrier for the subsurface fluids outflux. This is referred to as the top of geopressure (TOG). Studying many subsurface petrophysical properties behavior led to the updated conclusion of subdividing most of subsurface clastic into four main pressure compartments [3,4). This is because the change in porosity and consequently pore pressure directly impacts the rock sonic velocity, electric resistivity, and density. Figure 1 exhibits, in a nut-shell, the relationship between 15 million years of compartmentalized sedimentary sequence, velocity (sonic ∆t) profile drift due to compaction, pressure in psi, and the designated four zones (after shaker, 2019) [5]. The compaction drift of the data (sonic/resistivity etc.) in zone B [4,5] follows an exponential trend. The deeper extension of this trend is used to estimate the pore pressure (Figure 1).

Figure 1: Exhibits P-D conceptual cartoon plot represents a 15 million years four zones (A,B,C,D) of high sea stand shale beds interbedded with reservoir sand beds . Shale Velocity drift with depth reflects the pressure increase changes across the shale beds. CT is the compaction trend. Red arrows represent the Effective Stress [1,2]. The right display is in linear scale (exponential compaction trend), whereas the log display on the left is in logarithmic scale (linear compaction trend).

The following are some basic criteria for pressure prediction calculations:

  1. Predicting pore pressure (PP) is before drilling and calibrations/simulation are during and post drilling.
  2. Predicting PP is always in the section below the Top of Geopressure [1,2,5].
  3. Petrophysical data should be representing the clean shale/clay lithology only [1, 2 ].
  4. Normal hydrostatic pressure resides in the very shallow zone (A) only [3,4,5].
  5. The deeper extrapolation of zone B compaction trend is used only to calculate the Pore Pressure (PP) in zones C (top seal) and the compartmentalized geopressured zone D [2,5]. It is designated as CT instead of NCT by [5].
  6. Calibration of the predicting pressure’s model should not rely solely on the measured PP (MPP) in reservoir sands. Mud logs and drilling records should be collaborated in this process [5].

Pitfalls

Most of the pitfalls in calculation of subsurface pressure is driven by the lack of geological and geomechanical building blocks input in the prediction model. Since excess pressure generation and causes are the product of stressed water bearing formation, principal and minimum stresses vectors should be known in addition of the overburden gravity vector (Shaker,2024) [6]. Applying the old theorem that subsurface pressure profile is divided to two segments (Normal and Abnormal pressure zones) separated by the TOG can cause substantial miscalculations (Figure 2). This is because the misleading assumption of considering the compaction trend data set represents a normally pressured sequence (NCT). Compaction and expulsion of fluids by differential pressure is not normally hydrostatic pressure gradient [5]. Sequence stratigraphy also can be a guidance to geopressure compartmentalization and assessing sealing verses breaching reservoirs (Shaker, 2002) [7]. Calibrating and simulating the predicted pore pressure in the shale with the measured pp using the wireline tools in the reservoir can lead to substantial calculation errors. This due to the fact that most of the effective stress methods are designed for shale beds [1,2]. Figure 3 shows the prediction modeling blunder if the prediction model is enforced to follow the measured pressure value data. Utilizing pressure prediction software does not include the manipulation of extracting certain lithology or the flexibility of maneuvering the stresses vectors especially in salt basins and can be a main source of unintended pitfalls [6].

The symptoms of pitfalls are usually revealed on the interpreted pressure plots such as:

  1. Predicting pressure data trace (values) facing a porous/permeable lithology especially in reservoir sands/sandstones e.g. Bowers, 1995 [8], Ehsan M. et al. 2024 [9], and Merrell et al. 2014 [10].
  2. Extrapolating a Normal Compaction Trend (NCT) that covers the entire drilled lithological section four zones e.g. Berry et al. (2003) [11].
  3. Swaying and breaking the NCT to separate segments for the purpose of matching the predicted pp in the shale to the measured pp in the sand reservoirs e.g. Kuyken. and de Lange (1999) [12].
  4. Predicting pp in salt basins utilizing the Overburden as principal stress (S1), regardless the effect of salt – sediments differential stresses and salt buoyancy e.g. Shaker and Smith 2002 [13], Merrell et al. 2014 [10], and Zhang and Yin, 2017 [14].

In summary, geopressure prediction modeling is a product of multi-disciplinary geoscience and engineering fields of expertise. Therefore, collaboration between these different disciplines can improve and enhance forecasting and simulate a bonified subsurface pressure profile.

Figure 2: P-D plot showing correlation between Predicting seismic Velocity – Pressure model using the conventional NCT vs. CT/4 zones. The data using NCT shows ambiguous profile whereas prediction data using the CT/4 zone shows a bonified prediction with an agreement with the data extracted from nearby offset well.

 
 

Figure 3: Shows two P-D plots for the same deepwater well. On the left panel measured reservoir’s pp (RFT) only used as calibration tool for Predicting PP in shale (PPP shale). Note the mismatch between the circle and arrow zones. On the right panel is the right prediction method that using the shale only for prediction modeling.

Acknowledgement

Special Thanks to Kate Mariana of the research open world for facilitate publishing this short article.

References

  1. Terzaghi K, Peck RB (1948) Soil mechanics in engineering practice. John Wiley & Sons, Inc. N.Y., Pg: 566.
  2. Eaton BE (1975) The equation for geopressure prediction from well logs. Society of Petroleum Engineers of AIME paper #SPE 5544, 11.
  3. Shaker SS (2015a) A new approach to pore pressure predictions: Generation, expulsion, and retention trio—Case histories from the Gulf of Mexico. Gulf Coast Association of Geological Societies Transactions. 65: 323-s337.
  4. Shaker SS (2015b) Velocity for pore pressure modeling. Society of Exploration Geophysicist Annual Meeting, 1797-1801.
  5. Shaker SS (2019) Pore Pressure Prediction Before and During Drilling Applying the Four Zones Method, Offshore Technology Conference, OTC-29517-MS.
  6. Shaker SS (2024) Pore and fracture pressures prediction — A new geomechanic approach in deepwater salt overthrusts: Case histories from the Gulf of Mexico, Interpretation. 12: T501-T521,
  7. Shaker SS (2002) Predicted vs. Measured Pore Pressure: Pitfalls and Perceptions, Offshore Technology Conference, Houston Tx., OTC 14073.
  8. Bowers GL (1995) Pore pressure estimation from velocity data: accounting for overpressure mechanisms besides undercompaction. Society of Petroleum Engineers Paper SPE–27488, PA, Pg: 7.
  9. Ehsan M, Manzoor U, Chen R, Hussain M, Abdelrahman K, Radwan AE, Ullah J, Iftikhar MK, Arshad F, Pore pressure prediction based on conventional well logs and seismic data using an advanced machine learning approach, Journal of Rock Mechanics and Geotechnical Engineering, Pre – Print
  10. Merrell, M.P., Flemings, P.B., and Bowers, G.L.,2014, Subsalt pressure prediction in the Miocene Mad Dog field, Gulf of Mexico. AAPG Bulletin. 98: 315-340.
  11. Berry JR, McCormack N, Doyle E (2003) Overcoming pore-pressure challenges in deepwater exploration. OFFSHORE, Penn Well Corp., Reservoir Engineering.
  12. Kuyken CW, de Lange F (1999) Pore Pressure Prediction allows for tighter pressure gradient control, OFFSHORE, Penn Well Corp., Drilling Technology.
  13. Shaker SS, Smith M (2002) Pore Pressure Predictions in the Challenging Supra / Sub-Salt Exploration Plays in Deep Water, Gulf of Mexico. AAPG Search and Discovery Article #90007, presented at AAPG Annual Meeting.
  14. Zhang J, Yin S (2017) Fracture gradient prediction: an overview and an improved method. Springer, Pet Sci 14: 720-730.

Dysregulation of Cell-Mediated Immunity in Patients with Long ICU Stay Affected of Multiple Organ Dysfunction Syndrome and Multifocal Candidiasis

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DOI: 10.31038/IDT.2024523

Abstract

Introduction: The endogenous immunosuppression in critically ill patients with Multiple Organ Dysfunction Syndrome, requiring long-term ICU, can play an important role in the prognosis of Multifocal Candidiasis.

Method: Prospective study with control group (33 healthy volunteers) and study group (43 critically ill patients non neutropenic admitted to ICU with invasive mechanical ventilation), about cellular immunity in vitro, using Lymphoblastic Transformation Tests (LTT) stimulated with phytohemagglutinin (PHA). Simultaneously, the study group is followed up on appearance of Invasive Candidiasis and in vivo immunity study, using skin tests during the first week of ICU stay.

Results: There are significant statistical differences between the control group and the study group in relation to LTT PHA results. These differences are also found in the study group in relation to mortality and when skin tests are negative. When analysing the subgroup of patients with ICU stay longer than 12 days, there are statistically significant differences in LTT PHA when comparing the test performed in the 1st week in ICU in survivor patients with Invasive Candidiasis and the control group, but there are no differences with the 2nd determination. There are statistically significant differences when comparing the results of LTT PHA among patients with Invasive Candidiasis according to mortality in the 2nd determination, but these differences were not identified in the 1st week of ICU.

Conclusions: Both in vivo cellular immunity studies, using skin tests, and in vitro studies with the LTT PHA, help to establish a prognosis in critically ill patients with Multiple Organ Dysfunction Syndrome requiring long-term ICU care and in which a Multifocal Candidiasis has been identified. Monitoring of T-cell dysregulation and endogenous immunosuppression can help to identify patients who may benefit from new immunomodulatory therapeutic strategies.

Keywords

Cell-immunity, Multifocal candidiasis, Multiple organ dysfunction syndrome, Non-neutropenic, ICU

Introduction

In order to make Multifocal Candidiasis equivalent to Invasive Candidiasis, and thus initiate an early antifungal treatment to prevent death attributable to Candida spp. in non-neutropenic critical ICU patients who are neither neutropenic nor affected by AIDS [1,2], we analysed the factors that could contribute to the transformation from colonization of Candida spp. [2] to Multifocal Candidiasis and subsequent disseminated candidiasis. It is universally accepted that Candida spp. infections are linked to the antibiotic era [3]. The prolonged use of broad-spectrum antibiotics leads to an intestinal microbiological imbalance, promoting an excessive growth of candida in the intestinal lumen and thus facilitating the translocation of these fungi into the interior of the organism [4]. As an example, in the observations that were made, it was very common that, after treating with antibiotics treatment with broad-spectrum antibiotics for a sepsis due to Pseudomonas spp., if the patient survived and remained in critical condition, it was very common to detect the appearance of an invasive candidiasis. However, in these ICU patients who remain in a critical condition for a prolonged period of time (more than 7 days), other factors related to ICU treatment and monitoring can be identified, which should be considered as facilitators of Multifocal Candidiasis [5]:

  • Gastrointestinal dysfunction: Malnutrition and/or absence of enteral nutrition due to intestinal paresis; breakdown of the intestinal physiological barrier; treatment with parenteral nutrition, gastric protectors and prolonged nasogastric tube
  • Prolonged vesical catheterization
  • Prolonged arterial or venous catheter
  • Mechanical ventilation more than 7 days
  • Hemofiltration techniques

In some cases, this list can also be added treatment with prolonged treatment or high doses of corticosteroids (30% in these patients) [6]. It is at this point where it is questioned whether there may be in these patients an immunodeficiency acquired in the ICU and that this may play a role as important as the prolonged antibiotic treatment.

Methods and Materials

Prospective Study with Control Group

Study Group (43 patients): Patients over 18 years old and middle ages of 61 ± 10,8 (37-86), admitted to ICU with invasive mechanical ventilation without history of neutropenia or AIDS, and need for mechanical ventilation for more than 48h. There was no informed consent from the patients because their level of consciousness on admission to the ICU does not allow it. Family members have been informed about participation in the study, its risks and that this participation will not modify any of the protocols required in patient care throughout their stay in the ICU. Only the patients with the consent of their relatives have been included. Mortality rate of this study population was 46%. In 12 cases the UCI stay was lesser than 12 days with 42% mortality rate, and in 31 cases UCI stay was more than 12 days with a 48% mortality rate. The diseases that were the reason for ICU admission, grouped as follows were:

  • Chronic Obstructive Pulmonary Disease with respiratory failure requiring invasive mechanical ventilation prior to ICU admission (15 cases)
  • Postoperatory complicated cardiac or abdominal surgery with more than 24 h. of mechanical ventilation (12 cases)
  • Patients with Tetanus disease with invasive mechanical ventilation (13 cases).

Control Group (33 healthy volunteers): Group formed by subjects who have been informed prior to the acceptance of being included in this study.

Immunity Cellular Evaluation

  1. In vivo immunity study using skin tests, only in the study group. The test is considered positive if they present, at least in one of the puncture sites, an induration greater than 5 mm in diameter against the selected antigens (Streptokinase- Streptodornase 1/4, Candidin 1/500, P.P.D. 1/1000, Toxoplasmin). The test is performed during the first week of ICU stay and is not repeated at any other time.
  2. In vitro immunity study using Lymphoblastic Transformation Tests (LTT), lymphocyte extraction is performed in both Technique: An adjustment of lymphocytes to 2×106 cells/ ml is performed. Three control cultures and three stimulated with phytohemagglutinin (PHA) are grown for 72 h. at 37°C, with aerobic culture and 5% CO2. After this time, the slide extension and May-Grünwald, Giemsa staining is performed. Once the staining is done, the percentage of blasts over the total population of lymphocytes is calculated. This test, in the study group, is performed during the first 6 days of stay in the ICU and then every 12-17 days until discharge from the ICU.

Diagnosis of Multifocal Candidiasis

From the inclusion of the patients in the study group, cultures to identify Candida spp. are carried out once a week, until the ICU is discharged, to identify Candida spp. in five foci:

  1. Blood cultures
  2. Respiratory secretions from tracheobronquial aspiration
  3. Urinary cultures from closed circuit bladder catheterization. Positive only with more than 5000 colonies/ml.
  4. Simultaneous presence of positive cultures in the pharyngeal swab and in the gastric aspirate obtained through the nasogastric tube
  5. Other foci. Positive cultures from drainages or wound exudates

Candida colonization is defined when this study demonstrates the presence of Candida spp. in only one of these foci and the blood culture is negative. Candida multifocality or Invasive Candidiasis is defined when positive cultures are identified simultaneously in two or more of these foci and/or positive blood cultures.

Variables and Statistical Analysis Methodology

The following variables were analysed: ICU stay, ICU outcome, presence or absence of Candida spp. multifocality, presence of positive skin tests and results of LTT stimulated with PHA. Data were analysed using the SPSS statistical program. Categorical variables were compared among groups with Chi-square or Fisher exact test as appropriate. Continuous variables were analysed with the Student’s t or Mann-Whitney U test when the distributions departed from normality and described as mean or median (standard deviation or variance). Statistical significance was established at p value < 0.05 on two-tailed testing. For statistical analysis, patients were grouped according to mortality (D: dead/S: survivors) or the presence of positive skin tests (P: positive/N: negative).

Results

No significant differences were detected between the number of days spent in the ICU and mortality (42% mortality in stays of less than 13 days vs. 48% in stays of more than 12 days, X2 = 0.16). Significant differences were detected between patients according to whether or not they had positive skin tests and mortality (mortality of 27.8% in patients with positive skin tests vs. 78.3% with negative skin tests, X2 = 10.45 (p = 0.001)) and between presence of multifocal candidiasis and mortality (30% mortality in patients with colonization vs. 73.9% in patients with multifocality, X2 = 8.29 (p = 0.004)). These differences are more significant if only the group with stays longer than 12 days is analysed (10% mortality in patients with colonization vs. 71.4% in patients with multifocalities, X2 = 10.24 (p = 0.001)). There were no significant differences between positive skin tests and the presence of Multifocal Candidiasis (50% of patients with colonization had negative skin tests vs. 60.9% of patients with multifocality, X2 = 0.48).

Related on the LTT PHA done:

  1. There are significant statistical differences between the control group and the study group, in relation to mortality (LTT PHA control group (group C) = 39.25% (6.79), LTT PHA study group survivors (group S) = 29.22% (11.67), LTT PHA study group dead (group D) = 22.89% (13.11). Statistical differences between group C and group S: t = 3.86 (65 d.f.), p = 4×10-4; between group C and group D: t = 6.29 (65 d.f.), p =6×10-8; between group S and group D: t = 2.45 (66 d.f.), p = 0.002). Statistically significant differences are not found when comparing the results of the LTT PHA of the first determination between group S and group D (LTT PHA group S = 27.29% (10.15), LTT PHA group D = 22.68% (12.33), t = 1.49 (34 d.f.), p = 0.131).
  2. There were significant statistical differences between the control group and the study groups, whether the skin tests were positive or negative (LTT PHA control group (group C) = 39, 25% (6.79), LTT PHA study group with positive skin tests (group P) = 30.03% (12.16), LTT PHA study group with negative skin tests (group N) = 21.52% (12.41). Statistical differences between group C and group P: t = 3.8 (66 d.f.), p = 0.001; between group C and group N: t = 6.98 (60 d.f.), p = 5×10-9; between group P and group N: t = 3.39 (62 d.f.), p = 0.002). There were significant statistical differences when comparing the results of LTT PHA of 2nd determination between group P and group N (LTT PHA group P = 30, 07% (10.73) and LTT PHA group N = 19,28% (10,59), t = 2,13 (18 d.f.), p = 0.025). Statistically significant differences are not found when comparing the results of the LTT PHA of the 1st determination between group P and group N (LTT PHA group P = 29.68% (8.27) and LTT PHA group N = 22.53% (13.05), t = 1.5 (34 d.f.), p = 0.129).
  3. When analysing the subgroup of patients with ICU stay of more than 12 days, there were significant statistical differences between the control group and the study group according to survivors with colonization (1st study LTT PHA: t = 3,8 (39 d.f.), p = 0,001; 2nd study LTT PHA: t = 3,461 (35 d.f.), p = 0.002) and dead with multifocality for Candida spp (1st study LTT PHA: t = 5,82 (41 d.f.), p = 1,3×10-6; 2nd study LTT PHA: t = 6,09 (43 d.f.), p = 4,5×10-7). There were only significant statistical differences between the control group and the study group according to survivors with multifocality for Candida spp in the 1st study LTT PHA (1st study LTT PHA: t = 3,957 (37 d.f.), p = 0,0005; 2nd study LTT PHA: t = 0,603 (35 d.f.), p = 0,329). There were only significant statistical differences between dead with multifocality for Candida spp. and survivors with multifocality for Candida spp. in the 2nd study LTT PHA (1st study LTT PHA: t = 0,751 (14 d.f.), p = 0,291; 2nd study LTT PHA: t = 3,764 (14 d.f.), p = 0.002) (Table 1). There were no deaths in patients with candida colonization of more than 12 days.

Table 1: LTT PHA results, 1st determination and 2nd determination, comparing control group with patients staying in the ICU for more than 12 days in the study group, collected according to mortality and presence or not of Multifocal Candidiasis.

 Characteristics of the groups LTT PHA 1st determination

LTT PHA 2nd determination

Number of cases

 Median Variance Number of tests Median

Variance
Control Group Healthy volunteers

33

 39.25% 46 33 39.25% 46

Study Group with days of UCI stay > 12

Survivors and Candida spp colonization

 8 27.69% 104.62 4 26.70%

30.99
Survivors and multifocal candidiasis

6

 25.75% 111.35 4 37% 54.67

Dead and multifocal candidiasis

 10 20.75% 166.17 12 20.78%

163.54

Discussion

Critical illness is defined by presence of altered organ function in acutely ill patients such that homeostasis cannot be maintained without medical intervention in ICU, such as mechanical ventilation, vasoactive support for hemodynamic, renal replacement therapy, and so on [7]. The Sequential Organ Failure Assessment (SOFA) score is a simple method of assessing and monitoring organ dysfunction in critically ill patients [8,9]. The prognostic value of skin tests in critically ill patients has been observed for years [10]. There is also evidence of the prognostic value of Multifocal Candidiasis in these non- neutropenic critically ill patients. The detection of Multifocal or Invasive Candidiasis [2] can be an indicator of acquired immunosuppression in ICU patients affected by a Multiple Organ Dysfunction Syndrome [10]. In the patients of the study, the need for mechanical ventilation for more than 24 h. together with continuous sedation, before identifying Multifocal Candidiasis, indicates that their SOFA score is higher than 5. This fact confirms that the selected patients are admitted to the ICU with a Multiple Organ Dysfunction Syndrome. The LTT PHA carried out in this study demonstrate that the cellular immunity of non- neutropenic patients requiring mechanical ventilation for more than 48 h. presented significantly lower values than in a control group of non-neutropenic subjects. These results confirm the existence of an immunological response known as endogenous immunosuppression [11] in non-neutropenic critically ill patients. At the same time, these statistically significant differences disappear in the 2nd determination of the LTT PHA in critically ill patients with an ICU stay of more than 12 days and who survive. Therefore, improvement of this T-cell dysregulation [12] will condition ICU outcome in patients who remain in critical condition for more than 12 days. This observation raises the possibility that quantitative and qualitative kinetic monitoring of T-lymphocytes in ICU patients may help to identify those who may benefit from new immunomodulatory therapeutic strategies [13]. A possibility of this monitoring can be found following the proposal of the REALIST score [14].

Conclusions

Both in vivo cellular immunity studies, using skin tests, and in vitro studies with the LTT PHA help to establish a prognosis in critically ill patients requiring long-term ICU care and in which a Multifocal Candidiasis has been identified. Monitoring of T-cell dysregulation and endogenous immunosuppression can help to identify patients who may benefit from new immunomodulatory therapeutic strategies.

Aknowledgements

Thank you to Torres Rodriguez JM. MD PhD, who served as scientific advisor.

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