Molecular Point-of-Care Assay Development: Design and Considerations.

Molecular diagnostic point-of-care (MDx POC) testing is gaining momentum and is increasingly important for infectious disease detection and monitoring, as well as other diagnostic areas such as oncology. Molecular testing has traditionally required high-complexity laboratories. Laboratory testing complexity is determined by utilizing the Clinical Laboratory Improvement Amendments of 1988 (CLIA) Categorization Criteria scorecard, utilizing seven criteria that are scored on a scale of one to three. Previously, most commercially available point-of-care (POC) tests use other analytes and technologies that were not found to be highly complex by the CLIA scoring system. However, during the COVID-19 pandemic, MDx POC testing became much more prominent. Utilization during the COVID-19 pandemic has demonstrated that MDx POC testing applications can have outstanding advantages compared to available non-molecular POC diagnostic tests. This article introduces MDx POC testing to students, technologists, researchers, and others, providing a general algorithm for MDx POC test development. This algorithm is an introductory, step-by-step decision tree for defining a molecular POC diagnostic device meeting the functional requirements for a desired application. The technical considerations driving the decision-making include nucleic acid selection method (DNA, RNA), extraction methods, sample preparation, number of targets, amplification technology, and detection method. The scope of this article includes neither higher-order multiplexing, nor quantitative molecular analysis. This article covers key application considerations, such as sensitivity, specificity, turnaround time, and shipping/storage requirements. This article provides an overall understanding of the best resources and practices to use when developing a MDx POC assay that may be a helpful resource for readers without extensive molecular testing experience as well as for those who are already familiar with molecular testing who want to increase MDx availability at the POC. © 2024 Wiley Periodicals LLC.

ß-Thalassemia and Diabetes Mellitus: Current State and Future Directions.

ß-Thalassemia major is a congenital hemoglobin disorder that requires regular blood transfusion. The disease is often associated with iron overload and diabetes mellitus, among other complications. Pancreatic iron overload in ß-thalassemia patients disrupts ß-cell function and insulin secretion and induces insulin resistance. Several risk factors, including family history of diabetes, sedentary lifestyle, obesity, gender, and advanced age increase the risk of diabetes in ß-thalassemia patients. Precautionary measures such as blood glucose monitoring, anti-diabetic medications, and healthy living in ß-thalassemia patients notwithstanding, the prevalence of diabetes in ß-thalassemia patients continues to rise. This review aims to address the relationship between ß-thalassemia and diabetes in an attempt to understand how the pathology and management of ß-thalassemia precipitate diabetes mellitus. The possible employment of surrogate biomarkers for early prediction and intervention is discussed. More work is still needed to better understand the molecular mechanism(s) underlying the link between ß-thalassemia and diabetes and to identify novel prognostic and therapeutic targets.

Detection of Genes Encoding Microbial Surface Component Recognizing Adhesive Matrix Molecules in Methicillin-Resistant Staphylococcus aureus Isolated from Pyoderma Patients.

Pyoderma is a common skin infection predominantly caused by Staphylococcus aureus. In addition to methicillin resistance, this pathogen is resistant to many other antibiotics, which ultimately limits the available treatment options. Therefore, the present study aimed to compare the antibiotic-resistance pattern, to detect the mecA gene and the genes encoding microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) in S. aureus isolates. A total of 116 strains were isolated from patients suffering with pyoderma. Disk diffusion assay was opted to perform antimicrobial susceptibility testing of the isolates. Out of the isolates tested, 23-42.2% strains appeared susceptible to benzylpenicillin, cefoxitin, ciprofloxacin and erythromycin. While linezolid was found to be the most effective anti-staphylococcal drug, followed by rifampin, chloramphenicol, clindamycin, gentamicin and ceftaroline. Out of 116 isolates, 73 (62.93%) were methicillin-resistant S. aureus (MRSA). Statistically significant (p ≤ 0.05) differences in antibiotic resistance patterns between MRSA and methicillin-susceptible S. aureus (MSSA) were found. A significant association of resistance to ceftaroline, rifampin, tetracycline, ciprofloxacin, clindamycin, trimethoprim-sulfamethoxazole and chloramphenicol was found in MRSA. However, no significant difference was observed between MRSA and MSSA for resistance against gentamicin, erythromycin or linezolid. All cefoxitin-resistant S. aureus, nonetheless, were positive for the mecA gene. femA was found in all the MRSA isolates. Among other virulence markers, bbp and fnbB were found in all the isolates, while can (98.3%), clfA and fnbA (99.1%) were present predominately in MRSA. Thus, this study offers an understanding of antibiotic resistance MSCRAMMs, mecA, and femA gene patterns in locally isolated strains of S. aureus.

Resistance to Some New Drugs and Prevalence of ESBL- and MBL-Producing Enterobacteriaceae Uropathogens Isolated from Diabetic Patients.

Diabetes is a leading non-communicable disease and a risk factor for relapsing infections. The current study was aimed at investigating the prevalence and antibiotic susceptibility of carbapenem-resistant (CR) uropathogens of the family Enterobacteriaceae in diabetic patients. The data of 910 bacterial isolates was collected from diagnostic laboratories during January 2018 to December 2018. The bacterial isolates were identified using traditional methods including colonial characteristics, biochemical tests, and API (20E). Antimicrobial susceptibility and phenotypic characterization of ESBL, MBLs, and KPC was determined by utilizing CLSI recommended methods. The phenotypically positive isolates were further analyzed for resistance-encoding genes by manual PCR and Check-MDR CT103XL microarray. Susceptibility to colistin and cefiderocol was tested in accordance with CLSI guidelines. The data revealed that most of the patients were suffering from type 2 diabetes for a duration of more than a year and with uncontrolled blood sugar levels. Escherichia coli and Klebsiella pneumoniae were the most frequently encountered pathogens, followed by Enterobacter cloacae and Proteus mirabilis. More than 50% of the isolates showed resistance to 22 antibiotics, with the highest resistance (>80%) against tetracycline, ampicillin, and cefazolin. The uropathogens showed less resistance to non-ß-lactam antibiotics, including amikacin, fosfomycin, and nitrofurantoin. In the phenotypic assays, 495 (54.3%) isolates were found to be ESBL producers, while ESBL-TEM and -PER were the most prevalent ESBL types. The resistance to carbapenems was slightly less (250; 27.5%) than ESBL producers, yet more common amongst E. coli isolates. MBL production was a common feature in carbapenem-resistant isolates (71.2%); genotypic characterization also validated this trend. The isolates were found to be sensitive against the new drugs, cefiderocol and eravacycline. with 7−28% resistance, except for P. mirabilis which had 100% resistance against eravacycline. This study concludes that a few types of ESBL and carbapenemases are common in the uropathogens isolated from the diabetic patients, and antibiotic stewardship programs need to be revisited, particularly to cure UTIs in diabetic patients.

Sofosbuvir/Ledipasvir for Treatment of Chronic Hepatitis C Infection in Adult Patients with ß- Thalassemia Major: A Real-Life Study.

BACKGROUND & AIMS: Patients with thalassemia have a lifelong need for blood transfusion, which makes them more risky to hepatitis C virus (HCV). Iron overload and chronic HCV are considered risk factors for patients with thalassemia to develop liver insults. The aim of the present study is to investigate the safety and efficacy of sofosbuvir/ledipasvir in the treatment of chronic HCV infection in Egyptian adult patients with ß- thalassemia major. METHODS: A retrospective study included 53 patients with ß-thalassemia major with chronic HCV treated with sofosbuvir (400 mg) and ledipasvir (90 mg) as a single pill fixed-dose combination once daily for 12 weeks. The effectiveness of the treatment was assessed by the sustained virologic response (SVR) at 12 weeks after the end of the treatment. RESULTS: SVR was achieved in 96.23% of patients. 47.17% of patients had minor side effects. There was a significant reduction in ALT, AST, and serum ferritin 12 weeks post-therapy. There was an insignificant change in hemoglobin level or blood transfusion requirement 12 weeks posttherapy. There was no change in iron chelators doses throughout the study period. CONCLUSION: Sofosbuvir/ledipasvir regimen seems to be safe and highly effective in the treatment of chronic HCV in patients with ß-thalassemia major.

Clopidogrel combats neuroinflammation and enhances learning behavior and memory in a rat model of Alzheimer's disease.

BACKGROUND AND AIM: Alzheimer's disease (AD) is a progressive neurodegenerative disease. Multiple molecular mechanisms have been employed in its pathogenesis such as Amyloid ß (Aß) formation, tau protein hyperphosphorylation, reduced acetylcholine (ACh) level, and neuroinflammation. This study aimed to assess the possible neuroprotective effect of clopidogrel in AD model induced by aluminum chloride (AlCl3) in rats. METHODS: Sixty adult male Sprague-Dawley rats were divided into four different groups: Control, AlCl3, AlCl3 + donepezil, and AlCl3 + Clopidogrel. AlCl3 and the drugs were given orally once/day for 42 days. The spatial learning and memory and recognition memory were evaluated using Morris Water Maze (MWM) and Novel Object Recognition (NOR) tests, respectively. After euthanasia, hippocampal acetylcholinesterase (AChE) activity, tumor necrosis factor-alpha (TNF-α), and interleukin-1ß (IL-1ß) levels were biochemically assessed. Moreover, amyloid precursor protein (APP) mRNA gene expression was analyzed in the hippocampi of all rats. Histopathology for amyloid plaques was done. RESULTS: Clopidogrel co-treatment significantly ameliorated the cognitive deficits induced by AlCl3 in rats. Besides, clopidogrel significantly reduced AChE activity, TNF-α and IL-1ß concentrations, and APP mRNA gene expression in the hippocampi of rats compared to AlCl3 rats. The decrease of hippocampal TNF-α and IL-1ß concentrations by clopidogrel was significant compared to donepezil co-treated rats. Clopidogrel co-treatment lessened amyloid plaque deposition in the hippocampal tissues of rats compared to AlCl3 rats. CONCLUSION: These findings demonstrate that clopidogrel could alleviate learning and memory deficit induced by AlCl3 in rats and significantly reduced AChE activity. The neuroprotective outcome of clopidogrel might be assigned to its anti-inflammatory effect.

In vivo studies of the effect of PPQ-6, a quinoline-based agent against Schistosoma mansoni in mice.

Schistosomiasis is still a public health problem. Praziquantel is the only drug available for treatment of all forms of human schistosomiasis. Although praziquantel is an effective drug against all species of human schistosomes, concerns about resistance have been raised, especially in endemic areas. A hybrid compound containing several pharmacophore within a single molecule is a promising strategy. Here, we described the anti-schistosomal effect of 4-(2-Chloroquinolin-3-yl)-2-oxo-6-(p-tolyl)-1,2-dihydropyridine-3-carbonitrile (PPQ-6), a hybrid drug based on quinoline and pyridine. PPQ-6 was given as two regimens (20 or 40 mg/kg). In both regimens, PPQ-6 significantly reduced liver and spleen indices, nitric oxide production, tissue egg load, hepatic granuloma size and count, immature eggs and total worm burden especially females. Our findings suggested that PPQ-6 is a promising anti-schistosomal agent; however more research is needed to elucidate its mechanism of action and report its activity on juvenile schistosomes and other species of human schistosomes.

Biological evaluation of newly synthesized quinoline-based compound PPQ-8 in acute and chronic toxoplasmosis: An experimental study.

Toxoplasma gondii is a widely distributed protozoan parasite, which affects worm-blooded animals including human. The commonest chemotherapeutics used for treatment of symptomatic toxoplasmosis have numerous adverse effects. Thus there is an eminent need to develop new therapeutic agents. Here we described the therapeutic efficacy of 4-(2-chloroquinolin-3-yl)-6-(2,5-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (PPQ-8); a quinoline-related compound in a mouse model of acute and chronic toxoplasmosis. In acute infection, PPQ-8 decreased the parasite load in liver and spleen with amelioration of the hepatic and splenic pathology. In addition, recovered tachyzoites showed distorted shapes, reduced sizes, irregularities, surface protrusions, erosions and peeling besides apical region distortion when seen by scanning electron microscopy. In chronic toxoplasmosis, PPQ-8 produced degeneration and reduction of the brain cysts without stimulating a damaging inflammatory response within the brain. In both models acute and chronic, PPQ-8 prolonged the survival time of mice. These findings hold promise for the development of a novel anti-toxoplasmosis drug using PPQ-8, but further in vivo studies should be carried out to elucidate PPQ-8 mechanism of action and to report its efficacy in combination with other anti-toxoplasmosis agents.

A compilation of antimicrobial susceptibility data from a network of 13 Lebanese hospitals reflecting the national situation during 2015-2016.

Background: There is a lack of official national antimicrobial resistance (AMR) data in Lebanon. Individual hospitals generate their own antibiotic susceptibility data in the form of yearly pamphlets. Methods: In this study, antibiotic susceptibility data from 13 hospitals distributed across different governorates of Lebanon were collected to conduct a compilation-based surveillance of AMR in Lebanon for the years 2015-2016. The findings were compared with those of a previous nationwide study in this country conducted between 2011 and 2013 as well as with similar data obtained from the 2015 and 2016 European surveillance reports of AMR. To provide a clear presentation of the AMR situation, mean percent susceptibility of different antibiotic-microbe combinations was calculated. Results: During 2015-2016, the percent susceptibility of Enterobacteriaceae to third-generation cephalosporins and to carbapenems was 59 and 97%, respectively. Among Pseudomonas aeruginosa and Acinetobacter spp., carbapenem susceptibility reached 70 and 12%, respectively. Among Gram positive organisms, the percent susceptibility to methicillin in Staphylococcus aureus was 72%, that to vancomycin in Enterococcus spp. was 98% and that to penicillin in Streptococcus pneumoniae was 75%. Compared with results of 2011-2013, there was an overall trend of decreased susceptibility of bacteria to the tested antibiotics, with a variation of 5 to 10%. The antibiotic susceptibility data from Lebanon were found to be comparable with those from Eastern and South-eastern European countries. Conclusion: This study highlights the need to establish a robust national AMR surveillance system that enables data from Lebanon to be included in global AMR maps.

Could E-Cadherin and CD10 Expression be Used to Differentiate Between Atypical Endometrial Hyperplasia and Endometrial Carcinoma?

Endometrial carcinoma contributes to morbidity and mortality among female individuals worldwide. The role of E-cadherin expression, as an adhesion molecule, in endometrial carcinoma is controversial. Moreover, the role of CD10-expressing stromal cells in endometrial carcinoma is still unclear. The aim of this work was to evaluate E-cadherin and CD10 expression in normal endometrium, atypical endometrial hyperplasia, and endometrial carcinoma, and assess their role to differentiate atypical endometrial hyperplasia from endometrial carcinoma. The association of E-cadherin and CD10 expression with clinicopathologic parameters of endometrial carcinoma was also determined. This retrospective study was carried out on 80 cases including 36 cases of endometrial adenocarcinoma (all were of endometrioid type), 34 cases of atypical endometrial hyperplasia, and another 10 cases of normal endometrial tissue. Immunohistochemistry for E-cadherin and CD10 was conducted. The studied patients were in their sixth and seventh decades of life with a mean age of 60.97 yr. Most of the carcinoma cases (18 cases) were grade 1, 10 cases were grade 2, and only 2 cases were grade 3. With regard to International Federation of Gynecology and Obstetrics (FIGO) staging, 28 cases were stage I, and only 2 cases were stage II. E-cadherin in normal endometrial tissue and atypical hyperplastic endometrial tissue showed predominantly membranous homogenous reactivity, and CD10 was detected as membranocyptoplasmic staining. However, we noticed the subcellular change of E-cadherin reactivity to be heterogenous and predominantly membranocytoplasmic in endometrial carcinoma, whereas CD10 remained membranocytoplasmic. Concerning E-cadherin expression, there was a statistically significant relationship between E-cadherin expression, tumor grade and FIGO staging, whereas there was an insignificant relationship between E-cadherin expression and patients' age, specimen type, tumor gross pattern, and histopathologic types. With regard to CD10 expression, there was a statistically significant relation between CD10 expression and tumor grade and FIGO staging with insignificant relation with patients' age, tumor gross pattern, specimen type, and tumor histologic types (villoglandular vs. usual endometrial adenocarcinoma). There was also a highly statistically significant positive relationship between E-cadherin expression and CD10 expression. This study puts the spot light on their role in differentiating between atypical endometrial hyperplasia and endometrial carcinoma, which is often difficult.

Dengue fever in Lebanon: First confirmed case since 1945 and review from the region.

Lebanon yearly witnesses a high flux of expatriates and workers from Dengue virus (DENV) endemic regions. Multiple cases of Dengue fever have been documented at the American University of Beirut Medical Center (AUBMC) in travellers to endemic regions. Given the presence of the Aedes aegypti mosquito in Lebanon, introduction of DENV to the country is highly likely. We report a case of DENV infection in Lebanon diagnosed in April 2012 in a patient with no prior travel history. The patient presented with fever (39°C) and lower urinary tract symptoms and was initially diagnosed with culture negative prostatitis. He was started on empiric antibiotics but continued to have severe headache, diffuse myalgias, bone pain, and fatigue. He later developed a faint rash with leukopenia and thrombocytopenia. Extensive work-up was unrevealing. DENV IgM and IgG were positive suggesting acute infection. This is the first reported case since 1945 from Lebanon in a patient with no prior travel history.

CHALLENGES IN THE TREATMENT OF ACUTE FULMINANT HEPATITIS B: Case Report and Literature Review.

Acute hepatitis B is a serious cause of fulminant hepatic failure and subsequent mortality. No established guidelines are currently present for the treatment of this life threatening entity. Several therapeutic options were reported in the literature including the use of lamivudine as well as the more novel nucleoside analogue entecavir. We report an unfortunate case of fulminant hepatitis B that passed away despite intensive care unit management and treatment with entecavir in combination with steroids. Extensive review of the literature about various therapeutic approaches to manage fulminant hepatitis B was conducted. The aim of this report is to emphasize the need for larger more structured studies in order to improve the outcome of the treatment of this entity.

Osteomyelitis at two noncontiguous sites caused by Mycobacterium marinum in an immunocompetent host: case report and literature review.

Nontuberculous mycobacteria are rare causes of skin, soft tissue, and musculoskeletal infections. Mycobacterium marinum remains one of the most commonly encountered mycobacterial species in humans, causing superficial cutaneous as well as deep infections. We are reporting a case of M. marinum osteomyelitis involving two primary noncontiguous sites in an immunocompetent host, which was successfully treated with surgical drainage and antibiotic therapy.

Aspergillus mastoiditis in an immunocompetent patient: a case report and review of the literature.

Aspergillus mastoiditis usually occurs in immunocompromised patients. There are a few isolated reports in the literature involving immunocompetent patients. We hereby describe the case of an immunocompetent patient diagnosed with invasive Aspergillus mastoiditis, which was treated successfully, and review the literature pertaining to this condition. The common clinical presentations, putative pathophysiology, and recommended therapy are discussed.

Combination therapy for Gram-negative bacteria: what is the evidence?

The treatment of infections caused by multidrug-resistant Gram-negative bacteria is challenging given the limited options for effective therapy. Combination therapy has garnered great interest recently, with the goals of ensuring appropriate therapy with at least one active agent, and achieving synergistic activity among the anti-microbials used. In this review, we evaluate the data supporting the use of combination therapy against Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae, Acinetobacter species and Stenotrophomonas maltophilia. Various regimens have been tried with promising results; however, the data are mostly derived from in vitro synergy studies. While these reports suggest an advantage of combination therapy over monotherapy, clinical data are scarce, and are comprised of retrospective and a few prospective observational studies. Well-designed randomized trials are needed to better elucidate the efficacy of the various combination regimens. Until then, this review offers a critical appraisal of the published literature and provides recommendations based on the available evidence.

Stenting in Acute STEMI Intervention.

Stenting in acute myocardial infarction (AMI) has the benefits of achieving acute optimal angiographic results and correcting residual dissection to decrease the incidence of restenosis and reocclusion. Studies have shown that percutaneous transluminal coronary angioplasty for primary treatment after AMI is superior to thrombolytic therapy regarding the restoration of normal coronary blood flow. Coronary stenting improves initial success rates, decreases the incidence of abrupt closure, and is associated with a reduced rate of restenosis. In the presence of thrombus-containing lesions, coronary stenting constitutes an effective therapeutic strategy, either after failure of initial angioplasty or electively as the primary procedure.