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]


*Pneumocystis jirovecii* pneumonia (PJP) is one of the most prevalent opportunistic infections in immunocompromised conditions such as human immunodeficiency virus (HIV) infection, stem cell and solid organ transplantation, and malignancy. Because of the high mortality of the disease and potential adverse effects of the treatment regimens, early and accurate diagnosis of PJP is crucial and essential [@bib0105].

Since *P.* *jirovecii* is a non-culturable causative agent, many clinical microbiology laboratories rely on direct microscopic detection of the cysts of *P.* *jirovecii* from respiratory samples. However, this approach lacks sensitivity and might be particularly challenging in patients receiving anti-retroviral therapy with low organism load [@bib0105]. Because of the low sensitivity of microscopy and immunofluorescence (IFA) assay, several PCR technologies with higher sensitivity have been developed for detection of *P.* *jirovecii* from clinical samples [@bib0110], [@bib0115], [@bib0120].

Cytomegalovirus (CMV) is another common and important cause of opportunistic infection in immunocompromised hosts and is associated with high morbidity and mortality [@bib0125]. CMV is often detected from respiratory samples with other pathogenic agents, particularly *P.* *jirovecii,* in immunocompromised patients [@bib0130], [@bib0135]. CMV has immunomodulatory effects and inhibits the immune response generated against *P.* *jirovecii* and contributes to the delayed clearance of the *P.* *jirovecii* from patients [@bib0135].

The aim of this study was to evaluate the performance of the DiaSorin Molecular PJ-CMV PCR (DiaSorin Molecular LLC, Biotechnology Cypress, CA, USA) compared to direct IFA for *P.* *jirovecii* and assess co-occurrence of CMV and *P.* *jirovecii* infection in immunosuppressed patients.

This study was approved by the Institutional Review Board of Wake Forest Baptist Health.

Bronchoalveolar lavage (BAL) specimens from 35 HIV infected and 90 non-HIV infected immunocompromised patients with clinical suspicion of PJP were tested for *P.* *jirovecii* by direct IFA using the Monofluo *P.* *jirovecii* IFA test kit (MONOFLUO™ Bio-Rad, Laboratories, USA) according to the manufacturer\'s instructions. According to the clinical data reviewed, all patients evaluated were considered immunosuppressed and BAL samples were submitted for testing because patients met clinical and radiographic criteria for possible PJP and other infections had been ruled out. The non-HIV infected patients included patients with hemato-oncologic malignancies/stem cell or bone marrow transplant (*n*  = 38), solid organ transplant (*n*  = 17), and other immunosuppressed conditions (*n*  = 35). The remaining (surplus) specimen aliquots were stored at −70 °C until tested by PJ-CMV PCR.

Amplification of un-extracted PJ-CMV BAL samples for use with DiaSorin Molecular reagents was carried out in a total volume of 10 μl of sample without prior centrifugation. The primers allow detection of amplified *P.* *jirovecii* mtLSU and *CMV UL83* genes in the samples. Reactions contained: 2 μl of BAL samples or external controls, 0.2 μl PJ primer pair (Part number MOL9098), 0.4 μl CMV primer pair (Part number MOL9002), 4 μl TA master mix (Part number MOL9070), 0.2 μl Simplexa Extraction and Amplification Control DNA (Part number MOL9300), 0.2 μl Simplexa Extraction and Amplification Control primer pair (Part number MOL9300), and 1 μl of nuclease free water. Direct amplification reactions using DiaSorin Molecular PJ-CMV primer pairs were carried out on the 96-well Universal Disc using the LIAISON® MDX instrument with dye detection on for CRF610 (PJ), FAM (CMV), and Q670 (Internal control). Data collection and analysis were performed with LIAISON® MDX Studio software. The following cycling conditions were used: 1 cycle at 97̊C for 120 s followed by 40 cycles of 97̊C for 10 s with a ramp speed of 2̊C/s, and 60̊C for 30 s with a ramp speed of 2̊C/s with capture mode on. The test time of the PJ-CMV PCR assay from sample processing to final result is approximately 60 minutes. Extraction and amplification controls were used to detect PCR failure and/or inhibition. Positive and negative control samples were included in each run. Both CMV and PJ positive molecular controls were purchased from Exact Diagnostics (Exact Diagnostics, Fort Worth, TX). Standard procedures were used to obtain the analytical sensitivity of the test for the detection of *P.* *jirovecii* and CMV. Briefly, commercially available controls (CMV AD 169-Zeptometrix, Buffalo, NY, and *P.* *jirovecii* PCPP100-Exact Diagnostics, Fort Worth, TX) were chosen for the limit of detection (LOD) determination. Serial dilutions were prepared in a pooled BAL negative matrix ranging from 5000 copies/mL to 500 copies/mL for CMV and from 2500 copies/mL to 1000 copies/mL for *P.* *jirovecii*. LOD was defined as the minimum concentration with a detection rate of at least 95% by Probit Analyses. For the analytical specificity assessment, the manufacturer tested a panel of 31 microorganisms (Adenovirus 1, Inactive Influenza A, B, Parainfluenza 1, 2, 3, 4B, Rhinovirus 1A, Respiratory Syncytial Virus A, Metapneumovirus-9, Coronavirus 229E, Enterovirus 71, Herpes simplex virus 1, Cytomegalovirus, *Legionella pneumophila,* *L. longbeachae,* *Bordetella pertussis* ATCC 10380, *Klebsiella pneumoniae, Pseudomonas aeruginosa, Chlamydophila pneumoniae,* *Staphylococcus epidermidis, Streptococcus pyogenes, Cryptococcus neoformans, Mycobacterium tuberculosis,* *M.* *avium, Mycoplasma pneumoniae, M.* *hominis,* *Haemophilus influenzae, Candida catenulate, C.* *glabrata, C.* *parapsilosis,*) for *P.* *jirovecii* and 24 microorganisms (*Anaplasma phagocytophilum*, Adenovirus, Herpes simplex virus 1,2, Human herpes virus 6, 7, 8, BK Virus, Human immunodeficiency virus type 1, 2, Human T-lymphotropic Virus-1, Enterovirus, JC Virus, Epstein-Barr virus, Parvovirus B19, Hepatitis B virus, Hepatitis D virus, Rubella, Varicella zoster virus, *B.* *henselae*, *B.* *Quintana*, *Borrelia burgdorferi,* *Ehrlichia chaffeensis*, *Toxoplasma gondii*) for CMV known to cause pneumonia to confirm non-reactivity with *P.* *jirovecii* primers. The organisms were tested at a concentration of 1,000,000 CFU/mL for bacteria and 100,000 TCID50/ml for virus.

LOD was determined to be 2,063 ± 46 copies/mL or 103 copies/Rxn for CMV and 1,826 ± 44 copies/mL or 91 copies/Rxn for *P.* *jiroveci* in the current study*.* The assay yielded negative results for the panel of microorganisms other than the target organisms and thus no cross-reactivity was demonstrated. The results for the clinical samples showed that *P.* *jirovecii* was detected in 31.2% (39/125) and 40% (50/125) of the specimens using IFA and PJ-CMV PCR, respectively. When we compared the results obtained from HIV-infected and non-HIV infected patients, 16 (45.7%) of the 35 HIV infected and 34 (37.7%) of 90 non-HIV infected patients were positive for *P.* *jirovecii* by PJ-CMV PCR. Eleven of the samples positive by PJ-CMV PCR were negative by IFA. All 39 samples positive by IFA were also positive by PCR. There were no samples that were negative by PJ-CMV PCR and positive by IFA. The Ct for detection of PJ (number of cycles needed for a positive result) obtained with the PJ-CMV PCR was higher than 25 cycles (in 7 samples higher than 30) in the 11 samples negative by IFA suggesting that the PJ organism load was lower in the PJ-CMV PCR positive and IFA negative samples. Using IFA as a gold standard, the PJ-CMV PCR sensitivity, specificity, positive predictive value and negative predictive value for *P.* *jirovecii* were 100%, 87.2%, 78% and 100%, respectively ([Table 1](#tbl0005){ref-type="table"} ).Table 1Comparison of the *P.* *jirovecii* by direct immunofluorescence (IFA) and the PJ-CMV multiplex real-time PCR assay results (*N* = 125).Table 1IFA ResultPositiveNegativeTotalPJ-CMV multiplex real-time PCR ResultPositive391150Negative07575Total3986125

Of the 50 *P.* *jirovecii* positive samples, 18 (36%) were also positive for CMV by the PJ-CMV PCR. The co-infection rate was found to be 37.5% (6/16) and 35.2% (12/34) in HIV infected and non-HIV infected patients with immunosuppressed conditions, respectively.

There are several methods for detection of *P.* *jirovecii* from respiratory clinical samples including cytologic examination and IFA. Direct IFA test has been widely used and is still in first line for PJP diagnostic in association with pathogen DNA detection. These tests detect both the cystic and trophic forms of *P.* *jirovecii*. Although the direct IFA tests are more rapid to perform and easier to interpret than cytochemical stains, they might have false-negative results, especially in specimens from non-HIV infected patients since these tend to have fewer organisms present in the specimens [@bib0140], [@bib0145]. These tests also depend upon the skills and experience of the observer in terms of familiarity with the different morphologies of the organisms [@bib0150]. An overall 55.5% sensitivity and 98.6% specificity of direct IFA tests has been demonstrated in a meta-analysis article evaluating seven prospective studies [@bib0155]. Molecular techniques such as real-time PCR have been used in the diagnostic evaluation of PJP, which have better sensitivity than traditional cytologic stains and IFA [@bib0115], [@bib0120].

In the present study, we evaluated the performance of the PJ-CMV PCR assay on stored BAL samples against IFA for diagnosis of PJP. To our knowledge, this is the first evaluation of the PJ-CMV PCR assay using reagents from DiaSorin Molecular. The PJ-CMV PCR showed a higher sensitivity and specificity at diagnosing PJP in both HIV-infected and non-HIV infected immunocompromised patients compared to the direct IFA test. The higher sensitivity observed in this study is consistent with the results of previous studies on the detection of *P.* *jirovecii* [@bib0110], [@bib0160], [@bib0165]. A bivariate meta-analysis study by Lu et al. presented the diagnostic accuracy of *P.* *jirovecii* PCR techniques, with sensitivity ranging from 96% to 100% and specificity ranging from 87% to 93% [@bib0160]. In another review, reported sensitivities ranged from 82% to 100% and specificities ranged between 83% and 100% for PJP diagnosis using PCR assays [@bib0165]. A bivariate meta-analysis and systemic review by Fan et al. showed a very high diagnostic accuracy of PCR assays in BAL samples for the diagnosis of PJP with 98.3% sensitivity and 91.0% specificity evaluating 16 published studied [@bib0110].

Previous studies that have compared the performance of real-time PCR to IFA have also reported that a small number of samples were positive by real-time PCR but negative by IFA testing due to the higher sensitivity of the PCR [@bib0160], [@bib0170], [@bib0175]. In this study, eleven samples found positive by PCR were negative by IFA. All 11 of these with potentially false positive results compared to IFA had clinically proven PJP with clinical symptoms. These specimens had an overall higher cycle threshold value compared to samples positive by both methods, which became positive at less than 25 cycles. The specificity increased from 87.2 to 100% and the PPV increased from 78 to 100% once these eleven results were considered true-positive according to patients' clinical diagnostic criteria. One of the advantages of the PJ-CMV PCR assay is that it is designed as a closed system to reduce the potential carry-over contamination from run- to- run with amplicons causing false-positive results. Clinical samples are not open simultaneously during any step in the process. PJ-CMV PCR assay can be also used on other respiratory fluids like sputum or bronchial fluid.

CMV infection has immunomodulatory actions and aggravates the state of immunosuppression. It might lead to higher risk of opportunistic infections with other pathogens such as PJ and significantly influence on the outcome of the PJP infections [@bib0180], [@bib0185]. However, some studies have reported no significant effect of CMV co-infection on the outcome of PJP [@bib0125], [@bib0135], [@bib0190]. Although the clinical significance of concomitant CMV infection with PJP is poorly understood, some investigators advise that CMV should be monitored closely in PJP infected patients [@bib0125]. The PJ-CMV PCR assay detects both of these pathogens in a single sample, eliminating the need for a separate assay for CMV PCR testing in respiratory specimens. Previous studies have reported that 23.4--61.5% of PJP patients also exhibit co-infection with CMV [@bib0135], [@bib0185], [@bib0195], [@bib0200]. The percentage of CMV co-infection of this study was found to be 36% of patients, which is consistent with previous studies. There was no significant difference between the HIV infected and non-HIV infected patients in terms of the CMV co-infection.

There are a few limitations to this study. Our initial study question restricted the scope of this study and the data that we had access to. In particular, we compared the PJ-CMV PCR assay against direct IFA, the test routinely used at our laboratory, but not against another molecular test or against a quantitative test that could provide a specific organism load. In addition, although all samples analyzed were from patients meeting clinical criteria of PJP, we do not have further clinical data on the patients.

In summary, our findings demonstrate that the PJ-CMV multiplex real-time PCR assay showed higher sensitivity compared with direct IFA for detection of *P.* *jirovecii* in BAL samples and might be easily adapted for use in the clinical microbiology laboratories allowing the simultaneous detection of PJ and CMV in a single sample.

Disclosure of interest {#sec0005}
======================

The authors declare that they have no competing interest.

The Diasorin PJ and CMV PCR reagents used in this study were provided by DiaSorin Molecular LLC.