Ocular manifestations of liver disease: an important diagnostic aid.

PURPOSE: This review examined existing literature to determine various ocular manifestations of liver pathologies, with a focus on metabolic deficiencies as well as viral and immune liver conditions. METHODS: Recent data were compiled from PubMed from 2000 to 2020 using keywords that were relevant to the assessed pathologies. Ocular presentations of several liver pathologies were researched and then summarized in a comprehensive form. RESULTS: Several ocular manifestations of liver disease were related to vitamin A deficiency, as liver disease is associated with impaired vitamin A homeostasis. Alcoholic liver cirrhosis can result in vitamin A deficiency, presenting with Bitot spots, xerosis, and corneal necrosis. Congenital liver diseases such as mucopolysaccharidoses and peroxisomal disorders are also linked with ocular signs. Viral causes of liver disease have associations with conditions like retinal vasculitis, keratoconjunctivitis sicca, retinopathies, Mooren's ulcer, and Sjogren's syndrome. Autoimmune hepatitis has been linked to peripheral ulcerative keratitis and uveitis. CONCLUSIONS: Building strong associations between ocular and liver pathology will allow for early detection of such conditions, leading to the early implementation of management strategies. While this review outlines several of the existing connections between hepatic and ophthalmic disease, further research is needed in the area in order to strengthen these associations.

Enterovirus A71 infection-induced dry eye-like symptoms by damaging the lacrimal glands.

Purpose: To determine the effects of EV-A71 (Enterovirus A71) infection on ocular surface and its mechanism. Methods: AG6 mice aged two to three weeks were randomly divided into control and EV-A71 infected groups. Slit-lamp observation, fluorescein staining, and phenol red thread test were used to assess symptoms of ocular surface at 4 dpi (days post infection). The pathological changes of cornea and lacrimal gland were observed by H&E staining, PAS staining, TUNEL assay, IHC staining and qRT-PCR. Corneas and lacrimal glands from mice were obtained and processed for RNA sequencing analysis. Newly diagnosed HFMD patients caused by EV-A71 were recruited and ensured they met the inclusion criteria. Ocular surface parameters (TMH and NIKBUT) were measured using the OCULUS Keratograph 5M. Tear samples were taken to examine Cxcl1 and IL-6 levels through the ELISA method. Results: Mice studies revealed that EV-A71 infection caused tear film instability, decreased tear secretions, decreased in lacrimal gland size, and distinct goblet cell loss. It also resulted in increased large vacuoles within acinar cells and structural damage in lacrimal gland. Apart from minor damage to the epidermis, there was no obvious inflammatory changes or apoptosis in the cornea. However, there were significant inflammatory injury and apoptosis in the lacrimal gland. RNA-seq analysis showed IL-17 and NF-κB signaling pathways were activated in the lacrimal glands of mice infected with EV-A71. In HFMD patients, the THM was in a low range and NITBUT was significantly shorter than the control group by Oculus Keratograph 5M. ELISA assay showed a higher tear Cxcl1 and IL-6 level in them. Conclusion: EV-A71 infection affected lacrimal gland structure and function and induced dry eye-like symptoms.

A New Immortalized Human Lacrimal Gland Cell Line.

The lacrimal gland is crucial for maintaining ocular health by producing the aqueous component of the tear film, which hydrates and nourishes the ocular surface. Decreased production of this component results in dry eye disease, a condition affecting over 250 million people worldwide. However, the scarcity of primary human material for studying its underlying mechanisms and the absence of a cell model for human lacrimal gland epithelial cells present significant challenges. Here, we describe the generation of immortalized human lacrimal gland cell lines through the introduction of an SV40 antigen. We successfully isolated and characterized three cell clones from a female lacrimal gland donor, confirming their epithelial identity through genomic and protein analyses, including PCR, RNAseq, immunofluorescence and cultivation in a 3D spheroid model. Our findings represent a significant advancement, providing improved accessibility to investigate the molecular pathogenesis mechanisms of dry eye disease and potential therapeutic interventions. We identified the expression of typical epithelial cell marker genes and demonstrated the cells' capability to form 2D cell sheets and 3D spheroids. This establishment of immortalized human lacrimal gland cells with epithelial characteristics holds promise for future comprehensive studies, contributing to a deeper understanding of dry eye disease and its cellular mechanisms.

Assessment of tear film parameters in females with refractive errors using a single device: An observational study.

This observational study aimed to evaluate the use of a single portable device to assess the non-invasive tear break-up time (NITBUT), tear meniscus height (TMH), and lipid layer patterns (LLP) in young females with refractive errors (REs). The study was conducted at the College of Applied Medical Science (Female campus), Riyadh, Saudi Arabia between January 5, 2021 to May 15, 2021. Forty young females, with mean age of 23.0± 4.3 years with REs (-2.53 ± 2.05 D) and 40 females, mean age 23.8± 4.5 years with healthy eyes were recruited. The tests were administered in the following order: Ocular Surface Disease Index (OSDI), followed by NITBUT, TMH, and LLP. Significant differences (via Mann-Whitney U test) were noted in the median ocular surface disease index (OSDI; p˂0.001), NITBUT (p=0.035), TMH (p=0.009), and LLP (p˂0.001) scores between the study and control groups. Females with REs have significantly lower lipid layer, TMH, and NITBUT scores than those with healthy eyes.

Effects of Topical 0.05% Cyclosporine A on Dry Eye Symptoms and Parameters Following Small Incision Lenticule Extraction.

PURPOSE: To evaluate the effects of topical 0.05% cyclosporine A on Ocular Surface Disease Index (OSDI) score and ocular surface parameters after small incision lenticule extraction (SMILE) for myopia. METHODS: In this study, 151 patients who underwent SMILE were randomized into the control group (71 eyes) and the 0.05% cyclosporine A group (80 eyes). Both groups received standard treatment during the 1 month after SMILE. Over the next 3 months, The control group continued standard therapy (0.3% sodium hyaluronate) and the 0.05% cyclosporine A group received additional 0.05% cyclosporine A. OSDI total and subscale scores, non-invasive tear break-up time (NIBUT), tear lipid layer thickness (LLT), and tear meniscus height (TMH) were assessed preoperatively and postoperatively. RESULTS: Compared to baseline, the OSDI scores significantly increased in both groups (P < .001). The 0.05% cyclosporine A group exhibited lower OSDI total scores after administering 0.05% cyclosporine A versus the control group (P = .026). At 1 month of follow-up, NIBUT, LLT, and TMH values significantly decreased in both groups compared to baseline (P < .05). The 0.05% cyclosporine A group exhibited higher NIBUT, LLT, and TMH versus the control group, returning to preoperative values after 2 months. Overall, the OSDI total score and NIBUT values during follow-up were not significantly different between the two groups; however, the LLT and TMH values were significantly different between the two groups (P < .001 and .041, respectively) by repeated measures analysis of variance. CONCLUSIONS: Topical 0.05% cyclosporine A was effective in relieving subjective dry eye symptoms and maintaining ocular surface stability in the early postoperative period of SMILE. [J Refract Surg. 2024;40(4):e229-e238.].

Association between sleep quality and dry eye disease: a literature review and meta-analysis.

OBJECTIVE: The purpose of this article is to systematically review the association between dry eye and sleep quality. METHODS: PubMed, EMBASE, Cochrane, Web of Science, and grey literature databases were searched for observational studies published before April 2023. Meta-analysis was performed using STAT15 software. RESULTS: A total of 21 studies with 419,218 participants were included. The results showed that the dry eye subjects had a worse sleep quality than the healthy population, with poorer subjective sleep quality, longer sleep latency, and a higher risk of unhealthy sleep duration such as insufficient sleep or excessive sleep. The Pittsburgh Sleep Quality Index (PSQI) scores of the dry eye subjects were significantly higher than those of the control subjects (WMD = 1.78, 95%CI: 1.06, 2.50, P < 0.001). The dry eye subjects scored higher than the control subjects in sleep quality, sleep latency, and sleep disturbance in PSQI; there was no difference between the dry eye individuals and control subjects in sleep duration, sleep efficiency, daytime dysfunction, and sleep medication scores. The risk of sleep disorders in the dry eye subjects was significantly higher than that in the non-dry eye subjects (RR = 2.20, 95%CI: 1.78, 2.72, P < 0.001); the risk of insufficient sleep in the dry eye subjects was higher than that in the control subjects (RR = 3.76, 95%CI: 3.15, 4.48, P < 0.001), and the prevalence of excessive sleepiness in dry eye subjects was higher than that in the control subjects (RR = 5.53, 95%CI: 3.83, 7.18, P < 0.001). The ESS scores of the dry eye subjects were significantly higher than those of the control subjects (WMD = 3.02, 95%CI: 2.43, 3.60, P < 0.01). CONCLUSION: Our meta-analysis suggests that individuals with dry eye have a worse sleep quality than the healthy population, with poorer subjective sleep quality, longer sleep latency, and higher risk of unhealthy sleep duration such as insufficient sleep or excessive sleepiness.

New Korean Guidelines for the Diagnosis and Management of Dry Eye Disease.

New Korean guidelines for the diagnosis and management of dry eye disease were developed based on literature reviews by the Korean Dry Eye Guideline Establishment Committee, a previous dry eye guideline by Korean Corneal Disease Study Group, a survey of Korean Dry Eye Society (KDES) members, and KDES consensus meetings. The new definition of dry eye was also proposed by KDES regular members. The new definition by the regular members of the KDES is as follows: "Dry eye is a disease of the ocular surface characterized by tear film abnormalities and ocular symptoms." The combination of ocular symptoms and an unstable tear film (tear breakup time <7 seconds) was considered as essential components for the diagnosis of dry eye. Schirmer test and ocular surface staining were considered adjunctive diagnostic criteria. The treatment guidelines consisted of a simplified stepwise approach according to aqueous deficiency dominant, evaporation dominant, and altered tear distribution subtypes. New Korean guidelines can be used as a simple, valid, and accessible tool for the diagnosis and management of dry eye disease in clinical practice.

Computer vision syndrome and dry eye symptoms among breastfeeding women.

INTRODUCTION: Breastfeeding women are susceptible to musculoskeletal symptoms and hormonal changes that can affect the ocular surface. As exposure to visual display units (VDU) that is known to cause symptoms of dry eye and computer vision syndrome (CVS) is increasing worldwide, including among breastfeeding women, it is unknown whether this group of VDU users would experience CVS more than non-breastfeeding women. Therefore, this study aimed to investigate the association between breastfeeding status and symptoms of CVS and dry eye. MATERIALS AND METHODS: In this cross-sectional study, selfreported CVS and dry eye symptoms were compared between 80 breastfeeding and 72 non-breastfeeding VDU users. Two questionnaires were administered online, which were the CVS-Questionnaire (CVS-Q) and the Ocular Surface Disease Index (OSDI) questionnaire, to evaluate symptoms of CVS and dry eye, respectively. Mann-Whitney test was used to compare CVS and OSDI scores between groups, while correlations between the scores were analyzed using Spearman's test. RESULT: Results showed that OSDI scores were significantly higher in the non-breastfeeding group (U = 2263, z-score = - 2.276, p = 0.023), indicating more dry eye symptoms experienced by respondents in this group, while no significant group difference was found in terms of CVS scores (U = 2772, z-score = -0.400, p = 0.689). Additionally, no significant association was observed between breastfeeding status and severity of dry eye symptoms as well as CVS symptoms. CONCLUSION: This study reported the possible benefit of breastfeeding in reducing dry eye symptoms. The CVS symptoms found in the breastfeeding group were possibly due to VDU usage rather than caused by breastfeeding. Public education on preventive measures to reduce the occurrence of CVS symptoms and ocular dryness among VDU users is essential to improve the quality of life.

Role of Polyunsaturated Fatty Acids (PUFAs) and Eicosanoids on Dry Eye Symptoms and Signs.

Polyunsaturated fatty acids (PUFAs) generate pro- and anti-inflammatory eicosanoids via three different metabolic pathways. This study profiled tear PUFAs and their metabolites and examined the relationships with dry eye (DE) and meibomian gland dysfunction (MGD) symptoms and signs. A total of 40 individuals with normal eyelids and corneal anatomies were prospectively recruited. The symptoms and signs of DE and MGD were assessed, and tear samples (from the right eye) were analyzed by mass spectrometry. Mann-Whitney U tests assessed differences between medians; Spearman tests assessed correlations between continuous variables; and linear regression models assessed the impact of potential confounders. The median age was 63 years; 95% were male; 30% were White; and 85% were non-Hispanic. The symptoms of DE/MGD were not correlated with tear PUFAs and eicosanoids. DE signs (i.e., tear break-up time (TBUT) and Schirmer's) negatively correlated with anti-inflammatory eicosanoids (11,12-dihydroxyeicosatrienoic acid (11,12 DHET) and 14,15-dihydroxyicosatrienoic acid (14,15, DHET)). Corneal staining positively correlated with the anti-inflammatory PUFA, docosahexaenoic acid (DHA). MGD signs significantly associated with the pro-inflammatory eicosanoid 15-hydroxyeicosatetranoic acid (15-HETE) and DHA. Several relationships remained significant when potential confounders were considered. DE/MGD signs relate more to tear PUFAs and eicosanoids than symptoms. Understanding the impact of PUFA-related metabolic pathways in DE/MGD may provide targets for new therapeutic interventions.

IL-1ß induced down-regulation of miR-146a-5p promoted pyroptosis and apoptosis of corneal epithelial cell in dry eye disease through targeting STAT3.

AIM: To elaborate the underlying mechanisms by which IL-1ß promote progression of Dry eye disease(DED) through effect on pyroptosis and apoptosis of corneal epithelial cells(CECs). METHODS: 400 mOsM solutions were used to establish the DED model (hCECs- DED). RT-qPCR was performed to measure IL-1ß mRNA and miR-146a-5p in CECs. Western blotting was performed to measure STAT3, GSDMD, NLRP3, and Caspase-1 levels. Cell counting kit-8 assay was adopted to check cell viability. Apoptosis was detected by flow cytometry. ELISAs were performed to determine IL-18, IL-33 and LDH. The luciferase test detects targeting relationships. RESULTS: After treatment with 400 mOsM solution, cell viability decreased and apoptosis increased. Compared with hCECs, IL-1ß was increased and miR-146a-5p was decreased in hCECs-DED. At the same time, GSDMD, NLRP3, Caspase-1, IL-18, IL-33 and LDH were significantly higher in hCECs-DED than in hCECs, while IL-1ß silencing reversed this effect. In addition, IL-1ß negatively regulated miR-146a-5p. MiR-146a-5p mimics eliminated the inhibition of hCECs-DED pyroptosis and apoptosis caused by IL-1ß silencing. At the same time, miR-146a-5p reduced STAT3 levels in hCECs. CONCLUSION: Highly expressed IL-1ß promoted pyroptosis and apoptosis of hCECs- DED through downregulated miR-146a-5p and inhibited STAT3.

The efficacy of bandage contact lens in relieving the aggravation of dry eye disease after complicated cataract or/and IOL surgery.

PURPOSE: In the present study, we aimed to evaluate the efficacy of the bandage contact lens (BCLs) in the treatment of dry eye disease (DED) after complicated cataract or/and intraocular lens (IOL) surgery. METHODS: In this retrospective, single-centered, observational study, we collected data from 69 patients who underwent complicated cataract or/and IOL surgery. Of these, 35 cases wore their own BCLs immediately after the operation, while the other 34 cases did not have their own BCLs and were instead covered with gauze. The Ocular Surface Disease Index (OSDI) questionnaire, slit-lamp microscope examination, keratograph analysis, and Schirmer I test were measured at baseline, 1 week and 1 month postoperatively. RESULTS: In the BCL group, the score of the OSDI questionnaire was significantly decreased at 1 week and 1 month postoperatively compared with baseline levels (P = 0.000, collectively). Moreover, the fluorescein staining score of the BCL group was remarkably decreased 1-week and 1-month postoperatively compared with the non-BCL group (P = 0.000 and P = 0.000, respectively). Furthermore, the redness score of the BCL group was also better compared with the non-BCL group at 1 week and 1 month postoperatively (P = 0.014 and P = 0.004, respectively). CONCLUSIONS: Complicated cataract or/and IOL surgery would intensify the DED. Early application of BCLs postoperatively improved patients' comfort and alleviated dry eye-related symptoms and signs. Furthermore, this mechanism might involve the acceleration of corneal epithelial healing, the alleviation of ocular stress response and the stabilization of the tear film. TRIAL REGISTRATION: Trial registration ClinicalTrials, NCT04120389. Registered 10 October 2019-retrospectively registered.

Time-series analysis of the association between air pollution exposure and outpatient visits for dry eye disease: a case study in Zhengzhou, China.

Background: Dry eye disease (DED) is a prevalent ocular surface disease that significantly impacts patients' quality of life. The association between air pollution and the risk of dry eye disease remains uncertain. Methods: Data on outdoor air pollutants, meteorological information, and outpatient visits for DED were collected from July 1, 2014, to December 31, 2019. The relationship between ambient air pollutants and DED outpatient visits was analyzed using a generalized additive model with a Poisson distribution. Results: Among the 5,204 DED patients included in the study, 63.76% were female and 36.24% were male. The single-pollutant model revealed a significant association between a 10 µg/m3 increase in concentrations of fine-particulate matter with a median aerometric diameter of less than 10 µm (PM10), sulfur dioxide (SO2), nitrogen dioxide (NO2), and carbon monoxide (CO) and outpatient visits for DED. Fine-particulate matter with a median aerometric diameter of less than 2.5 µm (PM2.5) showed a significant association with DED outpatient visits in males and the 19-59 years age group. The strongest associations between air pollutants and outpatient visits were observed in male patients and during the cold season. Conclusion: The noteworthy correlation between air pollutants and DED outpatient visits can offer evidence for policy makers and underscore the significance of reinforcing environmental protection.

Utilization of Nitrogen-Doped Graphene Quantum Dots to Neutralize ROS and Modulate Intracellular Antioxidant Pathways to Improve Dry Eye Disease Therapy.

Purpose: Patients afflicted with dry eye disease (DED) experience significant discomfort. The underlying cause of DED is the excessive accumulation of ROS on the ocular surface. Here, we investigated the nitrogen doped-graphene quantum dots (NGQDs), known for their ROS-scavenging capabilities, as a treatment for DED. Methods: NGQDs were prepared by using citric acid and urea as precursors through hydrothermal method. The antioxidant abilities of NGQDs were evaluated through: scavenging the ROS both extracellular and intracellular, regulating the nuclear factor-erythroid 2-related factor (Nrf2) antioxidant pathway of human corneal epithelial cells (HCECs) and their transcription of inflammation related genes. Furthermore, NGQDs were modified by Arg-Gly-Asp-Ser (RGDS) peptides to obtain RGDS@NGQDs. In vivo, both the NGQDs and RGDS@NGQDs were suspended in 0.1% Pluronic F127 (w/v) and delivered as eye drops in the scopolamine hydrobromide-induced DED mouse model. Preclinical efficacy was compared to the healthy and DPBS treated DED mice. Results: These NGQDs demonstrated pronounced antioxidant properties, efficiently neutralizing free radicals and activating the intracellular Nrf2 pathway. In vitro studies revealed that treatment of H2O2-exposed HCECs with NGQDs induced a preservation in cell viability. Additionally, there was a reduction in the transcription of inflammation-associated genes. To prolong the corneal residence time of NGQDs, they were further modified with RGDS peptides and suspended in 0.1% Pluronic F127 (w/v) to create RGDS@NGQDs F127 eye drops. RGDS@NGQDs exhibited superior intracellular antioxidant activity even at low concentrations (10 µg/mL). Subsequent in vivo studies revealed that RGDS@NGQDs F127 eye drops notably mitigated the symptoms of DED mouse model, primarily by reducing ocular ROS levels. Conclusion: Our findings underscore the enhanced antioxidant benefits achieved by modifying GQDs through nitrogen doping and RGDS peptide tethering. Importantly, in a mouse model, our novel eye drops formulation effectively ameliorated DED symptoms, thereby representing a novel therapeutic pathway for DED management.

Deep learning-based fully automated grading system for dry eye disease severity.

There is an increasing need for an objective grading system to evaluate the severity of dry eye disease (DED). In this study, a fully automated deep learning-based system for the assessment of DED severity was developed. Corneal fluorescein staining (CFS) images of DED patients from one hospital for system development (n = 1400) and from another hospital for external validation (n = 94) were collected. Three experts graded the CFS images using NEI scale, and the median value was used as ground truth. The system was developed in three steps: (1) corneal segmentation, (2) CFS candidate region classification, and (3) estimation of NEI grades by CFS density map generation. Also, two images taken on different days in 50 eyes (100 images) were compared to evaluate the probability of improvement or deterioration. The Dice coefficient of the segmentation model was 0.962. The correlation between the system and the ground truth data was 0.868 (p<0.001) and 0.863 (p<0.001) for the internal and external validation datasets, respectively. The agreement rate for improvement or deterioration was 88% (44/50). The fully automated deep learning-based grading system for DED severity can evaluate the CFS score with high accuracy and thus may have potential for clinical application.

Comparison of comfort and tear stability with two novel silicone-hydrogel daily disposable contact lenses.

BACKGROUND: The surface wettability of soft contact lenses (SCLs) affects the wearing comfort. Verofilcon A is a daily disposable silicone hydrogel contact lens with SMARTSURFACE® technology, ensuring high surface wettability. PURPOSE: To evaluate tear stability on the surface of verofilcon A and narafilcon A SCLs and correlate these findings with clinical parameters and patients' discomfort. METHODS: Sixty-two SCL wearers (124 eyes) with SCL discomfort were randomly assigned to use narafilcon A (control SCL) and verofilcon A for 2 weeks each by crossover. The noninvasive tear break-up time (NIBUT) of the naked eye at baseline and pre-lens NIBUT (PL-NIBUT) of the SCL surface after 2 weeks of using each SCL were measured using the DR-1α® dry eye observation device. Corneal superficial punctate keratopathy (SPK) (0-3) and conjunctival hyperemia (0-3), and comfort level of SCL wear (1-10) were also evaluated. RESULTS: NIBUT and PL-NIBUT values were 4.6 ± 2.3 s for the naked eye, 6.6 ± 6.6 s for narafilcon A, and 11.3 ± 3.5 s for verofilcon A. verofilcon A had significantly higher PL-NIBUT than the naked eye and narafilcon A (p < 0.05). SPK (0.16 ± 0.48 vs. 0.00 ± 0.00, p < 0.01) and conjunctival hyperemia (1.15 ± 0.82 vs. 0.49 ± 0.50, p < 0.01) scores were lower when wearing verofilcon A than narafilcon A. The ocular comfort score of wearing SCLs was higher with verofilcon A than with narafilcon A (8.7 ± 1.8 vs. 9.8 ± 0.5, p < 0.01). The ocular comfort score for wearing verofilcon A was higher, regardless of the baseline NIBUT. CONCLUSION: The results of this study showed a consistent surface-wetting advantage of verofilcon A in patients with ocular discomfort. verofilcon A was used comfortably in patients with low NIBUT scores at baseline. The findings suggest that verofilcon A is recommended for SCL wearers experiencing SCL-related dry eye symptoms and discomfort.

Dry eye disease pathogenesis and clinical signs: searching for correspondence in the clinical practice.

BACKGROUND: Ocular surface alterations causing dry eye disease (DED) can be described as a vicious circle consisting of different consecutive stages. Among the factors involved, the ocular surface immune-inflammatory response has been established as a key player in the pathogenesis of the vicious circle of DED. Thus, the prompt recognition of the disruption of the immunoregulatory mechanisms is crucial for properly managing the ocular surface alterations. To increase awareness and knowledge of the identification and clinical interpretation of immunological mechanisms of dry eye in clinical practice, we present two clinical cases related to DED patients to provide a practical example of clinical examination application and interpretation of diagnostic parameters in daily practice. Moreover, a literature overview of the available clinical examinations to assess the immunological involvement in DED patients, with a particular focus on the correlation between diagnostic parameters and pathogenesis of clinical signs, is provided with an educational intent. CASE PRESENTATION: The presented clinical experiences suggested that in ocular surface pathologies, knowledge of the immune-inflammatory pathogenetic mechanisms underlying the observed clinical sign is of great help for understanding what is being observed in the patient and, consequently, for the choice of appropriate therapy. Literature evidence suggests that many different clinical examinations can be used to assess inflammation in DED patients, such as the assessment of hyperemia, staining of the ocular surface and measurement of hyperosmolarity and MMP-9 levels. The combination of impression cytology and flow cytometry to assess for markers of inflammation is considered the best technique to quantify the level of inflammation on the ocular surface, even if not always applicable in clinical practice. CONCLUSIONS: Literature evidence and clinical experiences suggest that basic diagnostic approaches (assessment of hyperemia, MMP-9 levels, and staining of the ocular surface with Lissamine green or fluorescein) represent useful tools to assess the inflammatory component of DED in everyday practice, providing a guide to establish the correct therapeutic strategy.