You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


I[NTRODUCTION]{.smallcaps} {#sec1-1}
==========================

Human immunodeficiency virus (HIV) infection is a significant risk factor for the occurrence of adrenocortical insufficiency (AI).\[[@ref1]\] Adrenal gland involvement has been documented in as many as two-thirds of patients with human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS) at postmortem examination.\[[@ref2]\] However, adrenal insufficiency is seldom diagnosed in clinical practice because symptoms do not appear until more than 80% of the gland has been destroyed.\[[@ref2]\]

Adrenal dysfunction can increase morbidity and mortality among patients with HIV infection.\[[@ref3]\] Adrenal insufficiency is a potentially modifiable comorbidity, especially as the biochemical abnormality (cortisol deficiency) is readily corrected by cortisol replacement.\[[@ref4][@ref5]\] AI should be suspected in patients with otherwise unexplained symptoms and signs compatible with adrenal insufficiency; such as anorexia, nausea, weight loss, and fatigue; and especially in patients with more specific manifestations of adrenal insufficiency, such as postural hypotension, hyponatremia, or hyperkalemia.\[[@ref3][@ref6]\]

Subclinical alterations in cortisol levels to frank primary adrenal insufficiency, using adrenocorticotropic hormone (ACTH) stimulation test, have been found in patients with HIV infection without symptoms or signs suggestive of adrenal insufficiency. In these patients, baseline cortisol levels are typically normal or elevated.\[[@ref5][@ref7][@ref8][@ref9][@ref10][@ref11]\] Some patients (8-14%) were found to have subnormal stimulation with ACTH in some studies.\[[@ref7][@ref8][@ref12][@ref13]\]

Although the adrenal gland is the most commonly affected endocrine organ in HIV,\[[@ref14]\] the clinical diagnosis of adrenal insufficiency is made difficult because HIV infection and AI share similar features.\[[@ref3]\] The classic clinical features and electrolyte derangements of AI have been shown to be poorly predictive of AI with regards to HIV infection.\[[@ref11]\] Symptoms such as weight loss, malaise, weakness, and diarrhea are common to both HIV infection and AI. Hyponatremia, a characteristic feature of AI, is common in HIV infection, where it may be due to inappropriate antidiuretic hormone secretion or gastrointestinal loss resulting from vomiting and diarrhea. Hyperkalemia, another characteristic biochemical feature of AI, can occur as a side effect of cotrimoxazole therapy in HIV patients. A deliberate search for AI will be required to identify AI in HIV patients. The standard for diagnosing AI is the dynamic testing of the adrenal gland using adrenocorticotropic hormone (ACTH); Synacthen^®^.

The progression of HIV infection is accompanied by immunodepression which is mainly due to the selective depletion of CD4 lymphocytes. In our practice, in a resource-poor setting, Synacthen^®^ is not readily available and when available, the cost is out of reach of the patients. The cost of cortisol assay is also expensive. However, CD4 count is done as part of the evaluation of persons with HIV infection to monitor the extent and progression of the disease. Whether CD4 count can be used to predict the presence or absence of AI in patients with HIV infection, has not been firmly established.

The main objective of this study was to determine the usefulness of CD4 count in predicting AI in persons with HIV infection. Other aims included documentation of the clinical and biochemical correlates of adrenal insufficiency in this group of patients.

M[ATERIALS AND]{.smallcaps} M[ETHODS]{.smallcaps} {#sec1-2}
=================================================

Consecutively presenting treatment naive persons with HIV infection who met the inclusion criteria were selected. The estimated sample size was 42. The sample size of 42 was arrived at by using the formula: *n* = Z^2^ pq/d^2^.\[[@ref15]\] For determination of sample size; P is the prevalence; the proportion of the target population estimated to have a particular characteristic. Estimated prevalence of HIV infection in Nigeria in 18-60 years age range is 5%.\[[@ref16]\] However, the prevalence of AI in persons with HIV infection was taken to be 50% since prevalence is not known in Nigeria.\[[@ref16]\] Z is the standard normal deviate which is 1.96. This corresponds to the 95% confidence interval. q is (1- p) and d which is the precision, is 0.15. Forty-two apparently, healthy non-HIV infected age and sex matched controls were selected. Making allowance for dropouts, (those who do not complete the study), 50 HIV infected patients were recruited for the study groups and 100 patients were recruited for the control group to increase the power of the study.

Persons who are pregnant, diabetic, and on drugs (e.g., steroid, cotrimoxazole, etc.) known to affect adrenocortical function were excluded from the study. Persons with HIV infection with concomitant pulmonary tuberculosis were excluded from the study. Persons with HIV infection who has had recent treatment with ketoconazole were excluded from the study. Children were also excluded from the study. Healthy volunteers, HIV negative, aged between 18 and 60 years who met the above criteria and consented to taking part in the study were recruited as control.

Of the 100 healthy volunteers (HIV negative) recruited as control for the determination of adrenocortical response to 1 μg of ACTH, 70 persons (35 males and 35 females) completed the exercise. Thirty persons declined further testing. The study group and the controls were recruited from the Lagos University Teaching Hospital (LUTH). Informed consent was obtained from all subjects and the study was approved by the LUTH Ethics and Research Committee.

The study groups were divided into batches of 10 subjects each. A data collection sheet, filled by the investigator, was used to obtain information from the subjects and controls. Information obtained from each participant included the biodata, presence of weakness, fatigue, cough, hemoptysis fever, weight loss, anorexia, nausea, vomiting, diarrhea, a history of glucocorticoid and/or antiretroviral drug use.

The subjects arrived on the assigned day at the laboratory, 60 min before the ACTH testing, after an overnight fast of 8-10 h. Physical examination including pulse rate and blood pressure in supine and erect position was performed. The anthropometric measurements (weight, height, and waist and hip circumference) were also taken. A 21-G canulla was inserted into a cubital vein and kept patent with heparinized saline. The subject then rested for 30 min after securing the venous access before samples were collected.

Low dose short Synacthen^®^ test was performed as follows: A baseline blood sample for cortisol, fasting plasma glucose (FPG), full blood count (FBC), erythrocyte sedimentation rate (ESR), CD4 count, and electrolytes were collected immediately before administration of ACTH. ACTH testing was conducted between 08.00 and 9.00 hour. After the samples had been taken, the subject received an intravenous bolus injection of 1 μg ACTH (Alliance Pharmaceuticals Ltd., Chippenham, Wiltshire SN15 2BB).

To prepare 1 μg of ACTH solution, 1 mL of ACTH solution was drawn from an ampule containing 250 μg/mL of ACTH. This was diluted with 249 mL of normal saline to yield a concentration of 1 μg/mL; 1 mL of this solution, containing 1 μg ACTH, was administered using 1 mL syringe, as bolus low dose injection. The remainder of the diluted ACTH solution was stored at 4°C in a refrigerator for subsequent use. After the bolus was administered, blood sample was drawn for cortisol level at 30 min. The samples were separated and transported on an ice slab to the laboratory where the plasma were stored at −20°C until assayed.

A normal basal cortisol, derived from 70 controls, was defined as a 0 min cortisol level of ≥145.1 nmol/L (mean-3SD). A normal response to 1 μg ACTH stimulation was defined as a 30 min cortisol level of ≥380.2 nmol/L (mean-3SD) and an increment from basal to stimulated cortisol level of ≥158.5nmol/L (mean -3SD). This was derived from 70 non-HIV infected controls. Using these values, AI in this study was defined as 30 min cortisol level \<380.2 nmol/L and increment from basal to stimulated cortisol level \<158.5 nmol/L. HIV infection was diagnosed if screened positive by enzyme-linked immunosorbent assay (ELISA method) and confirmed by immunoelectrotransference (Western blot).\[[@ref17]\] Postural hypotension was defined as difference between supine systolic blood pressure and erect systolic blood pressure \>20 mmHg.\[[@ref18]\] A diagnosis of hypoglycemia was made at plasma glucose level \<2.3 mmol/L.\[[@ref19]\] A diagnosis of hyponatremia was made at plasma sodium level \<135 mmol/L.\[[@ref20]\] A diagnosis of hyperkalemia was made at plasma potassium level \>5.0 mmol/L.\[[@ref20]\] A diagnosis of anemia was made at hemoglobin level \<12.0 g/dL.\[[@ref21]\] Low CD4 count refers to CD4 cells \<200/mm^3^.\[[@ref17]\]

Assay {#sec2-1}
-----

Serum cortisol levels were determined by an ELISA technique using the Diagnostic Automation Inc. cortisol assay method. It is a competitive immunoenzymatic colorimetric method for quantitative determination of cortisol concentration in serum. The respective intra assay and interassay coefficients of variation percentage (CV%) of 4.5 and 3.1% for serum cortisol were within the acceptable range of variation.

Statistical analysis {#sec2-2}
--------------------

Calculations and analysis were done using the SPSS 19.0 software. Continuous variables were expressed as means ± standard deviation (SD). Student\'s *t*-test was used for the comparison of means between two groups. Chi-square was used for comparison of proportions between two groups. Pearson\'s correlation coefficient analysis was used to determine associations between continuous data. The level of statistical significance was taken as *P* \< 0.05.

R[ESULTS]{.smallcaps} {#sec1-3}
=====================

A summary of the demographic and biochemical parameters are as shown in Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}. The mean basal cortisol (0-min) was 154.9 ± 27.2 nmol/L while the 30-min post ACTH stimulation cortisol level was 354.8 ± 19.9 nmol/L in HIV group. Using the normal cortisol values derived from healthy subjects, 15 (34.8%) persons with HIV infection had AI. Comparison of the standard recommended cutoff value (cortisol \< 500 nmol/L) with that used in this study showed that a higher proportion of person with HIV infection 38 out of 45 (83.4%) have AI \[[Table 3](#T3){ref-type="table"}\]. Of the patients with AI, seven (46.7%) had low CD4 count, while the remaining eight had CD4 count \> 200 cells/mm^3^ (χ^2^ = 1.246; *P* = 0.264). Of the 38 persons with AI using the standard recommended cutoff value, 21 (55.3%) had low CD4 count while 17 (44.7%) had CD4 count \> 200 cells/mm^3^ (χ^2^ = 1.111; *P* = 0.292). Low CD4 counts (CD4 \< 200 cells/mm^3^) was present in 25 (58.1%) of the patients with HIV infection. There was no significant correlation between basal cortisol levels and CD4 count in patients with HIV infection (r = -0.2, *P* = 0.198). There was no significant correlation between stimulated cortisol level and CD4 count in patients with HIV infection (r = -0.09, *P* = 0.516). In HIV patients with AI (15/43), negative correlation also exists between basal cortisol levels, stimulated cortisol level, and CD4 count however, this was not significant (r = -0.008, *P* = 0.976 and r = -0.039, *P* = 0.89, respectively). The risk of AI and CD4 count level is shown in [Table 4](#T4){ref-type="table"}. The proportion of persons with HIV infection with clinical features of AI is shown in [Table 5](#T5){ref-type="table"}. None of the patients with HIV had hyperpigmentation, postural hypotension, or hypoglycemia.

###### 

Demographic data in subjects with HIV and healthy subjects

![](IJEM-17-1012-g001)

###### 

Comparison of plasma biochemical variables in persons with HIV and healthy subjects

![](IJEM-17-1012-g002)

###### 

Comparison of a higher recommended cutoff value with that used in this study

![](IJEM-17-1012-g003)

###### 

Risk of AI and CD4 count

![](IJEM-17-1012-g004)

###### 

Clinical features of AI in HIV patients

![](IJEM-17-1012-g005)

D[ISCUSSION]{.smallcaps} {#sec1-4}
========================

Many investigators have evaluated adrenal function in critically ill patients by employing several approaches. Basal serum cortisol levels have been found to be elevated in patients with HIV, and therefore, are not reliable to exclude AI.\[[@ref3]\] The gold standard for the assessment of adrenal function is insulin tolerance test (ITT).\[[@ref22]\] The drawback in employing ITT to evaluate adrenal function is that it requires some degree of expertise and it is also unsafe in the setting of critical illness.\[[@ref22]\] Adrenocortical function is thus assessed using ACTH to stimulate the adrenal gland in the setting of critical illness. The standard dose (250 μg) of ACTH have been used to assess adrenocortical function.\[[@ref22]\] This however has been found to be the supraphysiological dose capable of inducing or mobilizing cortisol from other sources, thus giving a falsely normal test.\[[@ref22]\] This has led to investigators using 1 μg of ACTH to stimulate the adrenal gland. The 1 μg ACTH stimulation of the adrenal gland has been found to elicit maximal cortisol response comparable to the 250 μg ACTH test.\[[@ref23][@ref24]\] The 1 μg of synthetic ACTH has been suggested and used as a replacement for the standard 250 μg test.\[[@ref22][@ref25]\]

In this study, the mean basal cortisol value (154.9 nmol/L) and 30 min stimulated cortisol value (354.8 nmol/L) in persons with HIV was significantly lower than mean basal and 30 min stimulated cortisol levels in the healthy controls. The mean incremental rise (100 nmol/L) was also significantly lower in persons with HIV. Some investigators have used different diagnostic criteria for the diagnosis of AI as it relates to the population under study.\[[@ref26][@ref27][@ref28][@ref29]\] We also chose to define a diagnostic criteria for the population we studied. Using the diagnostic criteria for AI in this study, (peak cortisol \<380 nmol/L and increment \<158.5 nmol/L), we found that 15 (34.8%) persons out of 43 with HIV had AI. However, using serum cortisol level of 500 nmol/L suggested as the cutoff point for 30 min cortisol response,\[[@ref30]\] 38 (88.4%) out of 43 persons with HIV had subnormal response to ACTH test in this study \[[Table 3](#T3){ref-type="table"}\]. This might be an over estimation of the prevalence of adrenocortical failure in persons with HIV infection in the study, hence the use of a stringent diagnostic criteria in this study.

Our finding in this study is similar to the findings in previous studies.\[[@ref7][@ref8][@ref10][@ref13]\] The prevalence of 34.8% for AI is higher than 19% reported in a recent Ugandan study\[[@ref31]\] and 19% in a Brazilian study.\[[@ref32]\] In the Ugandan study, the basal cortisol level was used to diagnose AI. A higher prevalence in this study could be explained by the fact that stimulated cortisol value was used, whereas basal cortisol level was used in the Ugandan study. However, patients with HIV infection have been found to have normal basal cortisol level as demonstrated in this study.\[[@ref7][@ref8][@ref13]\] The abnormal response may be attributable to minimal degree of adrenal damage recognized in cases from autopsy studies.\[[@ref1][@ref33]\] The amount of adrenal gland tissue remaining functional, however, is apparently enough to provide a satisfactory glucocorticoid production in the basal state. In times of stress, adrenocortical response may not be adequate. AI might have been underdiagnosed in the Ugandan study.

The progression of HIV infection is accompanied by immunodepression which is mainly due to the selective depletion of CD4 lymphocytes. In a resource poor setting, ACTH stimulation of the adrenal gland poses a great challenge in terms of cost and availability of ACTH. One would have thought a measure of the HIV infection severity and progression (CD4 count) may be useful to predict AI. There exists a weak negative but not significant correlation between cortisol levels (basal and stimulated) and CD4 counts. This is similar to findings in other studies.\[[@ref34][@ref35][@ref36]\] In the subgroup of patient with AI, seven out of 15 (46.7%) have CD4 count below 200 cells/mm^3^ while the remaining eight (53.3%) have CD4 count above 200 cells/mm^3^. There is no significant difference between the two groups. The number of people with AI was lower in those with CD4 count \< 200 cells/mm^3^ than in those whose CD4 count was \> 200 cells/mm^3^. This, however, was not significant \[[Table 4](#T4){ref-type="table"}\].

The decrease in the number of CD4 lymphocytes is accompanied by an increase in serum cortisol. The associations between cortisol and a fall in the number of CD4 cells suggest that this steroid plays an important role in the normal function of the immune system through its effects on cytokine secretion and lymphocyte proliferation and activity.\[[@ref35][@ref36][@ref37]\] The increased serum cortisol concentration observed in people with HIV infection, most often without concomitant increase in circulating ACTH, suggests that the adrenal cortex is directly stimulated by other factors. Cytokines, such as interleukin-1 (IL-1) and interferon alpha (IFNα), acting indirectly or directly, could well modulate adrenal steroid production via their influence on the hypothalamic-pituitary-adrenal (HPA) axis.\[[@ref35][@ref36][@ref37]\] The increase in cortisol level while CD4 count is falling could also be explained by the fact that there is decreased cortisol catabolism caused by an abnormal fatty acid profile\[[@ref38]\] and alterations in the concentrations and binding properties of corticosteroid-binding globulin in HIV-infected patients.\[[@ref39]\]

None of the patients with HIV infection and impaired adrenal response to ACTH test (AI) had hyperpigmentation or postural drop in blood pressure. This was similar to the finding in a previous study.\[[@ref40]\] None of the person with HIV infection and AI had the classical hyperkalemia seen in adrenal insufficiency. This is similar to findings in other studies in other parts of Africa.\[[@ref40][@ref41]\] The mineralocorticoids are under the control of the renin-angiotensin system. This system is probably intact in people with HIV infection.

In conclusion, a CD4 count does not predict the presence or absence of AI. It cannot be used to diagnose adrenocortical function. A dynamic test using ACTH to stimulate the adrenal gland remains the acceptable standard.

L[IMITATIONS]{.smallcaps} {#sec1-5}
=========================

The preparation of 1μg ACTH could be affected by dilution error. It would have been ideal if there is commercially prepared 1 μg ACTH for stimulation of the adrenal glands. The use of lower cutoff value for the diagnosis of AI could have underestimated the proportion of persons with HIV infection with AI. These are possible limitations to our study.

**Source of Support:** Nil

**Conflict of Interest:** No