Mixed Aspergillosis and Mucormycosis Infections in Patients with COVID-19: Case Series and Literature Review.

BACKGROUND: Mucormycosis and aspergillosis are angioinvasive infections mainly occurring in immunocompromised patients. However, mixed infection with mucormycosis and aspergillosis in post-COVID-19 patients is rare. In this report, we will report four cases and comprehensively review the published literature on COVID-19 associated mixed infection of aspergillosis and mucormycosis. METHOD: Besides four of our cases, we searched for published articles using PubMed/MEDLINE, Scopus, and Web of Science databases from the beginning of 2020 until October 2023. RESULT: During the COVID-19 pandemic, we analyzed 52 cases (4 from our research and 48 from other studies). The most common underlying disease (59.6%) was diabetes mellitus. However, 19.2% of COVID-19 patients had no underlying condition. Interestingly, rhino-orbital-cerebral mucormycosis featured prominently in India and Iran, while other countries primarily reported a higher prevalence of pulmonary cases. CONCLUSION: In conclusion, this study highlights the presence of mixed aspergillosis and mucormycosis in COVID-19 patients who previously had common underlying diseases or even a healthy immune system. Therefore, managing COVID-19 patients should involve screening serum and respiratory samples using biomarkers to detect superinfections.

Transgenerational epigenetic effects imposed by neonicotinoid thiacloprid exposure.

Neonicotinoids are a widely used class of insecticides that are being applied in agricultural fields. We examined the capacity of a neonicotinoid, thiacloprid (thia), to induce transgenerational effects in male mice. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5-E15.5 using different doses. Testis sections were used for morphology analysis, ELISAs for testosterone level analysis, RT-qPCR and RNA-seq for gene expression analysis, MEDIP-seq and MEDIP-qPCR techniques for DNA methylation analysis, and Western blot for a protein analysis. The number of meiotic double-strand breaks and the number of incomplete synapsed chromosomes were higher in the thia 6-treated group of F3 males. Genome-wide analysis of DNA methylation in spermatozoa revealed that differentially methylated regions were found in all three generations at the promoters of germ cell reprogramming responsive genes and many superenhancers that are normally active in embryonic stem cells, testis, and brain. DNA methylation changes induced by thia exposure during embryonic period are preserved through several generations at important master regulator regions.

Molecular Biomarkers in Thyroid Eye Disease: A Literature Review.

PURPOSE: Thyroid eye disease (TED) is the most common extrathyroidal manifestation of Graves disease. Patients may be severely affected with eyelid retraction, exophthalmos, diplopia, pain, and threatened vision. Autoantibodies against thyroid-stimulating hormone receptor and insulin-like growth factor 1 receptor have shown associations with pathophysiological and clinical traits. Autoantibodies against thyroid-stimulating hormone receptor is in current clinical use as biomarker, but not with unambiguous diagnostic performance. A biomarker with high diagnostic accuracy and/or prognostic capability would be of immense value in diagnosing TED, especially in subclinical cases or when TED precedes the thyroid dysfunction. This article is a literature review on molecular biomarkers of TED. METHODS: A literature search was performed using PubMed and Embase. Studies on molecular biomarkers in blood, tear fluid, and urine were included in the review. RESULTS: Forty-six papers were included, of which 30, 14, and 2 studies on biomarkers in blood, tears, and urine, respectively. Fourteen of the papers evaluated the diagnostic performance of various biomarkers, 12 in blood and 2 in tears. Most studies evaluated single biomarkers, but 3 tested a panel of several markers. Except for autoantibodies against thyroid-stimulating hormone receptor, the reported diagnostic performances for the biomarkers were not confirmed in independent cohorts. In 32 studies, no or insufficient performance data were given, but the findings indicated involvement of various biologic mechanisms in TED including inflammation, oxidative stress, fibrosis, lipid metabolism, and ocular surface microflora. CONCLUSIONS: Currently, serum autoantibodies against thyroid-stimulating hormone receptor is the only molecular biomarker with clinical utility in patients with TED. Several potential biomarkers have been investigated, and particularly panels of multiple biomarkers in tears are promising. To improve patient care, biomarkers in TED should be studied further.

Statins in Graves Orbitopathy: A New Therapeutic Tool.

PURPOSE: Graves orbitopathy (GO) is the most common extrathyroidal manifestation of Graves disease. Although its pathogenesis is not fully elucidated, GO is commonly considered an autoimmune disease due to loss of self-tolerance against autoantigens shared by thyroid epithelial cells and orbital fibroblasts. High-dose intravenous glucocorticoids (ivGCs) are the most used treatment for moderate-to-severe, active GO, but the addition of other immunomodulating treatments can improve the efficacy of ivGCs. Among the various risk factors that can affect the occurrence of GO, cholesterol may be worthy of interest. Since 2015 the role of cholesterol and cholesterol-lowering medications has been investigated. The purpose of this review is to discuss this topic, thereby offering new therapeutic opportunities for patients with GO. METHODS: We searched PubMed for studies published between January 1, 1980 and June 1, 2023, using the search terms "Graves orbitopathy," "thyroid eye disease," "Graves ophthalmopathy," "thyroid ophthalmopathy," "thyroid-associated ophthalmopathy," "endocrine ophthalmopathy," "cholesterol," "lipids," "statins," "low-density lipoprotein," "atorvastatin," and "cholesterol-lowering drugs." Only English-language articles were included. RESULTS: A correlation between low-density lipoprotein cholesterol and the risk of GO development has been reported. Furthermore, low-density lipoprotein cholesterol has been proposed as a risk factor that can affect the course of GO and the response to ivGCs. The protective role of cholesterol-lowering medications in preventing GO has been also investigated. Statin treatment was found to have potential benefits in reducing the risk of GO in patients with Graves disease. Given these findings, measurement of low-density lipoprotein cholesterol and treatment of hypercholesterolemia in patients with moderate-to-severe, active GO may be considered before starting ivGCs administration. Recently, a randomized clinical trial aimed at investigating the effects of statins in GO suggested that the addition of oral atorvastatin to ivGCs improves the overall outcome of moderate-to-severe, active GO in hypercholesterolemic patients given ivGCs. CONCLUSIONS: Overall, statins seem to have a preventive and therapeutic role in moderate-to-severe active GO. Their efficacy can be related to cholesterol-lowering activity, pleiotropic actions, and interaction with methylprednisolone.

Dysthyroid Optic Neuropathy.

PURPOSE: Dysthyroid optic neuropathy (DON) is a sight-threatening complication of thyroid eye disease (TED). This review provides an overview of the epidemiology, pathogenesis, diagnosis, and current therapeutic options for DON. METHODS: A literature review. RESULTS: DON occurs in about 5% to 8% of TED patients. Compression of the optic nerve at the apex is the most widely accepted pathogenic mechanism. Excessive stretching of the nerve might play a role in a minority of cases. Increasing age, male gender, smoking, and diabetes mellitus have been identified as risk factors. Diagnosis of DON is based on a combination of ≥2 clinical findings, including decreased visual acuity, decreased color vision, relative afferent pupillary defect, visual field defects, or optic disc edema. Orbital imaging supports the diagnosis by confirming apical crowding or optic nerve stretching. DON should be promptly treated with high-dose intravenous glucocorticoids. Decompression surgery should be performed, but the response is incomplete. Radiotherapy might play a role in the prevention of DON development and may delay or avoid the need for surgery. The advent of new biologic-targeted agents provides an exciting new array of therapeutic options, though more research is needed to clarify the role of these medications in the management of DON. CONCLUSIONS: Even with appropriate management, DON can result in irreversible loss of visual function. Prompt diagnosis and management are pivotal and require a multidisciplinary approach. Methylprednisolone infusions still represent first-line therapy, and surgical decompression is performed in cases of treatment failure. Biologics may play a role in the future.

Intraoperative use of electrical impedance spectroscopy for adenoid cystic carcinoma of the lacrimal gland: A case report.

Key Clinical Message: Electrical Impedimetric Tumor Detection System is a novel and promising tool for fast intraoperative tumor delineation and accurate safe margin detection in orbital tumors. Abstract: Adenoid cystic carcinoma (ACC) is a rare malignant tumor of epithelial origin, typically arising from the salivary and lacrimal glands. ACC is notorious for recurrence and a high rate of morbidity and mortality despite therapy. We presented a 48-year-old male patient with lacrimal gland ACC of the right orbit who underwent radical tumor resection and adjuvant radiotherapy. We applied a new diagnostic method, the Electrical Impedimetric Tumor Detection System, during surgery and tested its performance to enhance the precision of tumor resection. Two months after surgery, he underwent external radiation of 58 Gy in 29 fractions. He showed no tumor recurrence or metastasis in the 1-year follow-up visits. ITDS showed a precision of tumor and margin detection consistent with histopathology results. This novel ITDS may be a reliable system for fast intraoperative tumor delineation and accurate, safe margin detection in orbital tumors.

COVID-19 Associated Rhino-Orbital-Cerebral Mucormycosis: Clinical Features, Antifungal Susceptibility, Management and Outcome in a Tertiary Hospital in Iran.

BACKGROUND: Despite the unprecedented surge in the incidence of mucormycosis in the COVID-19 era, the antifungal susceptibility patterns (ASPs) of COVID-19 associated mucormycosis (CAM) isolates have not been investigated so far and it is unclear if the high mortality rate associated with CAM is driven by decreased susceptibility of Mucorales to antifungal drugs. OBJECTIVES: To describe the clinical, mycological, outcome and in vitro ASPs of CAM cases and their etiologies from Iran. PATIENTS/METHODS: A prospective study from January 2020 to January 2022 at a referral tertiary hospital in Tehran, Iran was conducted for screening mucormycosis through histopathology and mycological methods. The identity of Mucorales isolates was revealed with ITS-panfungal PCR& sequencing and MALDI-TOF. The AS for amphotericin B, itraconazole, isavuconazole and posaconazole was cleared according to the EUCAST antifungal susceptibility testing protocol. RESULT: A total of 150 individuals were diagnosed with CAM. Males constituted 60.7% of the population. The mean age was 54.9 years. Diabetes was the leading risk factor (74.7%). The median interval between diagnosis of COVID-19 and CAM was 31 days. The recovery rate of culture was as low as 41.3% with Rhizopus arrhizus being identified as the dominant (60; 96.7%) agent. Amphotericin B (MIC50 = 0.5 µg/ml) demonstrated the highest potency against Mucorales. CONCLUSION: Majority of the cases had either diabetes, history of corticosteroid therapy or simultaneously both conditions. Accordingly, close monitoring of blood glucose should be considered. The indications for corticosteroids therapy are recommended to be optimized. Also, an anti Mucorales prophylaxis may be necessitated to be administrated in high risk individuals. Although amphotericin B was the most active agent, a higher rate of resistance to this antifungal was noted here in comparison with earlier studies on mucormycetes from non-CAM cases.

The Role of Estrogen Receptors in Health and Disease.

Many biological and physiological events, including growth, development, and metabolism of reproductive and non-reproductive tissues in men and women, are regulated by estrogens and estrogen receptors (ERs) [...].

Skull base osteomyelitis: A case report of Garcin syndrome due to mucormycosis in COVID pandemic.

Garcin syndrome is characterized by progressive unilateral multiple cranial nerve palsy without the presence of intracranial hypertension. In this case, we present a patient who experienced lower cranial nerve (CN 9-12) involvement attributed to post-mucormycosis osteomyelitis of the skull base. The osteomyelitis resulting from mucormycosis led to the development of Garcin syndrome, which manifested as progressive paralysis of the cranial nerves. It is important to recognize this rare complication and consider it in the differential diagnosis when evaluating patients with lower cranial nerve palsy following mucormycosis-related skull base osteomyelitis.

Erythropoietin Protects against Retinal Damage in A Rat Model of Optic Neuropathy via Glial Suppression.

OBJECTIVE: Traumatic optic neuropathy (TON) causes partial or complete blindness because death of irreplaceable retinal ganglion cells (RGCs). Neuroprotective functions of erythropoietin (EPO) in the nervous system have been considered by many studies investigating effectiveness of this cytokine in various retinal disease models. It has been found that changes in retinal neurons under conditions of glial cells are effective in vision loss, therefore, the present study hypothesized that EPO neuroprotective effect could be mediated through glial cells in TON model. MATERIALS AND METHODS: In this experiment study, 72 rats were assessed in the following groups: intact and optic nerve crush which received either the 4000 IU EPO or saline. Visual evoked potential and optomotor response and RGC number were assessed and regenerated axons evaluated by anterograde test. Cytokines gene expression changes were compared by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Density of astrocytes cells, assessed by fluorescence intensity, in addition, possible cytotoxic effect of EPO was measured on mouse astrocyte culture in vitro. RESULTS: in vitro data showed that EPO was not toxic for mouse astrocytes. Intravenous injection of EPO improved vision, in terms of visual behavioral tests. RGCs protection was more than two times in EPO, compared to the vehicle group. More regenerated axons were determined by anterograde tracing in the EPO group compared to the vehicle. Moreover, GFAP immunostaining showed while the intensity of reactive astrocytes was increased in injured retina, systemic EPO decreased it. In the treatment group, expression of GFAP was down-regulated, while CNTF was upregulated as assessed by qRT-PCR in the 60th day post-crush. CONCLUSION: Our study showed that systemic administration of EPO can protect degenerating RGCs. Indeed, exogenous EPO exerted neuroprotective and neurotrophic functions by reducing reactive astrocytic gliosis. Therefore, reduction of gliosis by EPO may be considered as therapeutic targets for TON.

A Basic Review on Estrogen Receptor Signaling Pathways in Breast Cancer.

Breast cancer is the most common cancer and the deadliest among women worldwide. Estrogen signaling is closely associated with hormone-dependent breast cancer (estrogen and progesterone receptor positive), which accounts for two-thirds of tumors. Hormone therapy using antiestrogens is the gold standard, but resistance to these treatments invariably occurs through various biological mechanisms, such as changes in estrogen receptor activity, mutations in the ESR1 gene, aberrant activation of the PI3K pathway or cell cycle dysregulations. All these factors have led to the development of new therapies, such as selective estrogen receptor degraders (SERDs), or combination therapies with cyclin-dependent kinases (CDK) 4/6 or PI3K inhibitors. Therefore, understanding the estrogen pathway is essential for the treatment and new drug development of hormone-dependent cancers. This mini-review summarizes current literature on the signalization, mechanisms of action and clinical implications of estrogen receptors in breast cancer.

Is obesity-induced ECM remodeling a prelude to the development of various diseases?

Due to the increasing incidence rate of obesity worldwide and the associated complications such as type 2 diabetes and cardiovascular diseases, research on the adipose tissue physiology and the role of the extracellular matrix (ECM) has gained tremendous attention. The ECM, one of the most crucial components in body tissues, undergoes remodeling and regeneration of its constituents to guarantee normal tissue function. There is a crosstalk between fat tissue and various body organs, including but not limited to the liver, heart, kidney, skeletal muscle, and so forth. These organs respond to fat tissue signals through changes in ECM, function, and their secretory products. Obesity can cause ECM remodeling, inflammation, fibrosis, insulin resistance, and disrupted metabolism in different organs. However, the mechanisms underlying the reciprocal communication between various organs during obesity are still not fully elucidated. Gaining a profound knowledge of ECM alterations during the progression of obesity will pave the way toward developing potential strategies to either circumvent pathological conditions or open an avenue to treat complications associated with obesity.

Codon adaptation by synonymous mutations impacts the functional properties of the estrogen receptor-alpha protein in breast cancer cells.

Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these cancers is to understand and overcome the mechanisms of endocrine resistance. Recently, two distinct translation programmes using specific transfer RNA (tRNA) repertoires and codon usage frequencies were evidenced during cell proliferation and differentiation. Considering the phenotype switch of cancer cells to more proliferating and less-differentiated states, we can speculate that the changes in the tRNA pool and codon usage that likely occur make the ERα coding sequence no longer adapted, impacting translational rate, co-translational folding and the resulting functional properties of the protein. To verify this hypothesis, we generated an ERα synonymous coding sequence whose codon usage was optimized to the frequencies observed in genes expressed specifically in proliferating cells and then investigated the functional properties of the encoded receptor. We demonstrate that such a codon adaptation restores ERα activities to levels observed in differentiated cells, including: (a) an enhanced contribution exerted by transactivation function 1 (AF1) in ERα transcriptional activity; (b) enhanced interactions with nuclear receptor corepressor 1 and 2 [NCoR1 and NCoR2 (also known as SMRT) respectively], promoting repressive capability; and (c) reduced interactions with SRC proto-oncogene, non-receptor tyrosine kinase (Src) and phosphoinositide 3-kinase (PI3K) p85 kinases, inhibiting MAPK and AKT signalling pathway.

Challenges and Pitfalls in the Management of Rhino-Orbital Mucormycosis in Ophthalmology: A Highlighted Problem in the COVID-19 Era.

Secondary infections in hospitalized and ill patients with coronavirus disease 2019 (COVID-19) are common. One of these life-threatening infectious diseases is rhino-orbital mucormycosis, which made an outbreak recently. This outbreak was mainly caused by the administration of high-dose corticosteroids in patients with COVID-19, especially those with diabetes mellitus. The increased incidence of rhino-orbital mucormycosis in the COVID-19 era presents different challenges for healthcare providers including ophthalmologists who are directly involved in disease management. We summarized the main challenges and recommendations for ophthalmologists on the management of rhino-orbital mucormycosis.

Mouse Degenerating Optic Axons Survived by Human Embryonic Stem Cell-Derived Neural Progenitor Cells.

Objective: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating axons and ultimately death of retinal ganglion cells (RGCs) that in most cases, are not curable by surgery or medication. Neuroprotective functions of different types of stem cells in the nervous system have been evaluated in many studies investigating the effectiveness of these cells in various retinal disease models. Neural progenitor cells (NPCs) secrete an assortment of trophic factors that are vital to the protection of the visual system. We aimed to assess the therapeutic potentials of NPCs in an ONC mouse model. Materials and Methods: In this experimental study, NPCs were produced using noggin and retinoic acid from human embryonic stem cells (hESCs). Fifty mice were divided into the following three groups: i. Intact , ii. Vehicle [optic nerve crush+Hank's balanced salt solution (HBSS)], and iii. Treatment (optic nerve crush+NPCs). The visual behavior of the mice was examined using the Visual Cliff test, and in terms of RGC numbers, they were assessed by Brn3a immunostaining and retrograde tracing using DiI injection. Results: Intravenous injection of 50,000 NPCs through visual cliff did not produce any visual improvement. However, our data suggest that the RGCs protection was more than two-times in NPCs compared to the vehicle group as examined by Brn3a staining and retrograde tracing. Conclusion: Our study indicated that intravenous injection of NPCs could protect RGCs probably mediated by trophic factors. Due to this ability and good manufacturing practices (GMP) grade production feasibility, NPCs may be used for optic nerve protection.

Interference with zinc homeostasis and oxidative stress induction as probable mechanisms for cadmium-induced embryo-toxicity in zebrafish.

The present study was conducted to provide new insights into the mechanisms that may be responsible for cadmium (Cd)-induced toxicity in zebrafish larvae as well as the role of the trace element zinc (Zn) in reversing Cd harmful effects. For this purpose, zebrafish eggs were exposed to Cd or/and Zn for 96 h. The effects on morphological aspect; mortality rate; Cd, Zn, and metallothionein (MT) levels; oxidative stress biomarkers; as well as molecular expression of some genes involved in Zn metabolism (Zn-MT, ZIP10, and ZnT1) and in antioxidant defense system (Cu/Zn-SOD, CAT and GPx) were examined. Our results showed that Cd toxicity was exerted, initially, by an interference with Zn metabolism. Thus, Cd was able to modify the expression of the corresponding genes so as to ensure its intracellular accumulation at the expense of Zn, causing its depletion. An oxidative stress was then generated, representing the second mode of Cd action which resulted in developmental anomalies and subsequently mortality. Interestingly, significant corrections have been noted following Zn supplementation based, essentially, on its ability to interact with the toxic metal. The increases of Zn bioavailability, the improvement of the oxidative status, as well as changes in Zn transporter expression profile are part of the protection mechanisms. The decrease of Cd-induced MTs after Zn supplement, both at the protein and the mRNA level, suggests that the protection provided by Zn is ensured through mechanisms not involving MT expression but which rather depend on the oxidative status.

Disease modifying drugs in idiopathic sclerosing orbital inflammatory syndrome.

PURPOSE: Idiopathic sclerosing orbital inflammatory syndrome (ISOIS) is a rare, progressive and hard to control disease. There is a deep gap of evidence regarding application of disease-modifying drugs (DMD) regimen as a potentially effective treatment for orbital inflammatory diseases. We aimed to report the results of using DMDs and discuss the concept of applying this modality of treatment in patients with ISOIS. METHODS: This was a prospective interventional case series conducted in a tertiary university-based hospital. Biopsy proven patients with active ISOIS were included. Systematic criteria were developed to define and measure disease activity and monitor response to treatment. A DMD regimen including an anti-tumor necrosis factor alpha (anti-TNF alpha) agent plus azathioprine and low-dose corticosteroids were used. Comprehensive ophthalmic, orbital and systemic assessments were performed during each visit. RESULTS: Five eligible patients with primary ISOIS were included. Mean age was 34.20 (SD = 13.33, range 19-53) years. Three had unilateral and two had bilateral involvement. Four had diffuse orbital involvement pattern and progressive worsening of visual functions, reduced extraocular motility and proptosis. In one patient the disease was localized to extraocular muscle and lacrimal gland. Disease activity was decreased and stabilized after DMDs regimen in all patients. Mean follow up was 32.80 (SD = 30.80, range: 12-86) months. CONCLUSION: Biologic DMD (b-DMD) including anti-TNF alpha, corticosteroid and azathioprine were effective in decreasing disease activity and could change course of the disease. This study supports the concept of using b-DMD regimen in treatment of ISOIS.

Pediatric Graves' orbitopathy: a multicentre study.

PURPOSE: Graves' orbitopathy (GO) is a rare condition in children often considered to be a less severe condition than at an older age. The aim of our study was to analyse if there are any factors that distinguish paediatric from adult GO in order to provide guidelines for assessing and managing paediatric GO. METHODS: Study design is a multicentre retrospective observational case series; 115 paediatric patients diagnosed with GO who visited our university medical centres in the Netherlands and Iran between 2003 and 2019 were submitted for complete ophthalmological examinations, serological testing and/or orbital imaging. Main outcome measures focussed on the natural course and clinical picture as well as medical and surgical treatment in paediatric GO. RESULTS: Clinical findings included proptosis (n = 97; 84.3%), eyelid retraction (n = 77; 67%) and diplopia (n = 13; 11.3%). Ninety-two patients (80%) presented with mild disease, 21 (18.3%) with moderate-severe disease and two (1.7%) with severe GO. Five patients (4.3%) underwent intravenous glucocorticoids and 25 patients underwent orbital decompression surgery. Strabismus surgery due to primary involvement of extraocular muscles was performed in two patients (1.7%). Overall, rehabilitative surgical treatment was planned in 31 patients (26.9%) with inactive disease. Two patients experienced reactivation of the disease. CONCLUSION: Despite the fact that paediatric and adult GO are considered two separate entities, they might be the same disease with two different clinical phenotypes. Paediatric GO population presents with a comparable clinical picture regarding both soft tissue involvement and proptosis, which may require surgical intervention. Proptosis was present in the majority of paediatric GO patients. Orbital decompression was performed in 21.7% of patients.

Key parameter optimization using multivariable linear model for the evaluation of the in vitro estrogenic activity assay in T47D cell lines (CXCL-test).

In comparison with analytical tools, bioassays provide higher sensitivity and more complex evaluation of environmental samples and are indispensable tools for monitoring increasing in anthropogenic pollution. Nevertheless, the disadvantage in cellular assays stems from the material variability used within the assays, and an interlaboratory adaptation does not usually lead to satisfactory test sensitivities. The aim of this study was to evaluate the influence of material variability on CXCL12 secretion by T47D cells, the outcome of the CXCL-test (estrogenic activity assay). For this purpose, the cell line sources, sera suppliers, experimental and seeding media, and the amount of cell/well were tested. The multivariable linear model (MLM), employed as an innovative approach in this field for parameter evaluation, identified that all the tested parameters had significant effects. Knowledge of the contributions of each parameter has permitted step-by-step optimization. The most beneficial approach was seeding 20,000 cells/well directly in treatment medium and using DMEM for the treatment. Great differences in both basal and maximal cytokine secretions among the three tested cell lines and different impacts of each serum were also observed. Altogether, both these biologically based and highly variable inputs were additionally assessed by MLM and a subsequent two-step evaluation, which revealed a lower variability and satisfactory reproducibility of the test. This analysis showed that not only parameter and procedure optimization but also the evaluation methodology must be considered from the perspective of interlaboratory method adaptation. This overall methodology could be applied to all bioanalytical methods for fast multiparameter and accurate analysis.