Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Minimal residual disease testing for classical Hodgkin lymphoma: A comprehensive review.

Classical Hodgkin lymphoma (cHL) is a common lymphoma that affects young patients. Fortunately, the disease is highly curable as it is susceptible to the currently available treatment modalities. Disease monitoring with Positron Emission Tomography and Computed Tomography (PET/ CT) is an integral part of managing these patients. PET guided protocols are currently used to adjust treatment according to the response. The pivotal idea behind the use of response-adapted approaches is to preserve efficacy while decreasing the toxicity. It also helps to intensify therapy in patients in need because of suboptimal response. However, imaging techniques are limited by their sensitivity and specificity. Minimal Residual Disease (MRD) assessment is a newly emerging concept in many hematologic malignancies. It utilizes various molecular techniques such as polymerase chain reaction (PCR), and next-generation sequencing (NGS) as well as flow cytometry, to detect disease traces. This review looks into MRD detection techniques, its current applications, and the evidence in the literature for its use in cHL.

Additional reporting of diffuse and homogeneous ROS-1 SP384 immunoreactivity enhances prediction of ROS1 fusion-positive non-small cell lung cancer.

OBJECTIVES: ROS-1 immunohistochemistry (IHC) is a common method for screening ROS1 fusion in the clinical management of non-small cell lung cancer. The interpretation criteria for ROS-1 SP384 IHC, however, remain unestablished. METHODS: Sixty-five non-small cell lung cancer cases underwent AmoyDx ROS1 fusion real-time polymerase chain reaction (PCR) study and ROS-1 SP384 IHC tests, which were retrieved for analysis. ROS-1 IHC tests were interpreted based on the established classifiers as well as the presence of diffuse homogeneous immunoreactivity. The diagnostic accuracies of these ROS-1 IHC interpretation methods were evaluated by comparing them with the ROS1 real-time PCR results. RESULTS: Previous ROS-1 IHC classifiers demonstrated high sensitivity for positive ROS1 real-time PCR results (100%), but they showed low specificities (25%-50%) and overall accuracies (58%-72%). In contrast, the diffuse homogeneous ROS-1 immunoreactivity predicted positive ROS1 real-time PCR results with much higher specificity (94%) and overall accuracy (95%), albeit with a slightly lower sensitivity (97%). Some cases that showed discrepancy between diffuse homogeneous ROS-1 immunoreactivity and real-time PCR results involved rare ROS1::LDLR fusion and suboptimal IHC staining. CONCLUSIONS: A 3-tier reporting system for ROS-1 SP384 IHC testing combining previous interpretation criteria and diffuse and homogeneous immunoreactivity may better predict ROS1 fusion status without decreasing specificity.

Role of WT1 in Measurable Residual Disease Follow-Up in the Post Allogeneic Stem Cell Transplant Setting.

Objectives: The Wilms' tumor gene 1 (WT1) plays a critical role in cell development and the regulation of essential genes involved in cell growth and metabolism. In the context of hematopoietic tumors, including acute myeloid leukemia (AML), WT1 has been identified as a potential marker for measurable residual disease (MRD) assessment. Relapse after allogeneic hematopoietic stem cell transplantation (allo-SCT) remains a significant challenge in AML treatment, highlighting the importance of MRD monitoring for risk stratification and treatment decisions. This study aimed to investigate the clinical significance of WT1 as a molecular marker for MRD and its correlation with chimerism in AML patients post-allo-SCT setting. Methods: We have included 58 patients with WT1-expression-positive acute myeloid leukemia (AML) who received allo-SCT in our center between 2016-2022. The exclusion criteria are as follows: not having WT1 polymerase chain reaction (PCR) measurement at diagnosis, not receiving allo-SCT, and not having a serial measurement of WT1 post-transplant. Pre- and post-transplant assessments were made with flow cytometry, WT1 PCR, and bone marrow morphological evaluations. Statistical analyses were carried out to explore correlations between WT1 levels, MRD markers, and chimerism post-transplantation. Results: We found that WT1 had a significant correlation with flow cytometry and bone marrow morphological evaluation, but not with chimerism. Interestingly, high WT1 expressors exhibited a more robust correlation with chimerism compared to the general cohort. The negative predictive value for post-allo-SCT relapse was 91.8% for the whole WT1 cohort; for high WT1 expressors, it was similar, at 87.5%. The negative predictive value for post-allo-SCT relapse was high for the whole WT1 cohort; for high WT1 expressors, it was similar. The WT1 MRD assay showed a high negative predictive value for post-allo-SCT relapse, consistent across both the entire cohort (91.8%) and high WT1 expressors (87.5%). Conclusions: WT1 expression levels may serve as a valuable ancillary marker in MRD assessment and relapse prediction post-allo-SCT in AML patients, particularly for those lacking specific fusion genes or mutations. However, further large-scale, controlled studies are needed to standardize WT1 MRD assays and establish clear guidelines for their clinical application.

Imatinib dose reduction after major molecular response in chronic-phase chronic myeloid leukemia.

BACKGROUND: In people with chronic-phase chronic myeloid leukemia (CML) receiving imatinib and achieving major molecular response (MMR), dose reduction may decrease adverse events but may be associated with a loss of molecular response. Whether digital droplet polymerase chain reaction (ddPCR) can identify persons in whom dose reduction might be unsuccessful is unknown. METHODS: Data from 716 consecutive subjects who achieved MMR after initial imatinib therapy (400 mg/day) were obtained. A total of 486 subjects remained on full-dose imatinib, whereas 230 subjects had their dose reduced to 300 or 200 mg/day. The outcomes of these cohorts were compared via landmark and propensity score matching analyses. RESULTS: Imatinib dose reduction showed no significant effect on the subsequent achievement of deeper molecular responses (4- and 4.5-log reductions in BCR::ABL1 transcripts; MR4 and MR4.5), maintenance of MMR, or attainment of therapy-free remission when compared with subjects without dose reduction. In subjects achieving MR4, however, the probability of maintaining MR4 (p = .002) was lower in the reduced-dose group. In multivariable analyses, failure to achieve MR4.5 as determined by ddPCR at the time of dose reduction was significantly associated with briefer MMR failure-free survival (FFS; hazard ratio [HR], 10.3; 95% confidence interval [CI], 1.3-82.9; p = .03) and MR4 FFS (HR, 6.8; 95% CI, 2.6-18.0; p < .001). CONCLUSIONS: Imatinib dose reduction after achieving MMR does not adversely affect response deepening or MMR maintenance in chronic-phase CML but compromises MR4 maintenance. The results of ddPCR may identify people who benefit from imatinib dose reduction.

Culture and other direct detection methods to diagnose human granulocytic anaplasmosis.

OBJECTIVES: We sought to assess the performance of 3 laboratory tests on blood specimens for direct detection of Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA), in patients tested at a single medical institution in New York State. METHODS: Direct tests included microscopic blood smear examination for intragranulocytic inclusions, polymerase chain reaction (PCR), and culture using the HL-60 cell line. The HGA cases testing positive by only 1 direct test were not included, unless HGA was confirmed by acute or convalescent serology using an indirect immunofluorescent assay. RESULTS: From 1997 to 2009, 71 patients with HGA were diagnosed by at least 1 of the 3 direct test methods. For the subgroup of 55 patients who were tested using all 3 methods, culture was positive for 90.9% (50/55) vs 81.8% (45/55) for PCR vs 63.6% (35/55) for blood smear (P =.002). Most cultures (79.3%) were detected as positive within 1 week of incubation. CONCLUSIONS: Although using culture to detect A phagocytophilum is likely not amenable for implementation in most hospital laboratories, in our experience, culture had the highest yield among the direct tests evaluated.

Streptococcus spp. isolated from bovine mastitis: Antimicrobial sensitivity studies and disagreement evaluation between routine phenotypic diagnosis and molecular identification.

Bovine mastitis poses a significant threat to global dairy production, resulting in substantial losses in milk production. Streptococcus bacteria, particularly Streptococcus uberis, Streptococcus agalactiae, and Streptococcus dysgalactiae, are commonly implicated in this condition. An accurate diagnosis is crucial for implementing effective treatment and minimizing its impact on production. This study examined 115 Streptococcus strains isolated from bovine mastitis cases in Uruguay using PCR for species identification. Additionally, the resistance to tetracycline, erythromycin, and penicillin was assessed in 81 of the bacterial strains under study. Significant disparities between phenotypic and genotypic detection were evident across all three species, with only 31% of strains identified phenotypically aligning with PCR results. Phenotypic prevalence indicated S. dysgalactiae as the most prevalent (44.35%), followed by S. uberis (24.34%) and S. agalactiae (6.09%). However, the genotypic identification revealed S. uberis as the most prevalent, followed by S. dysgalactiae, while S. agalactiae remained the least prevalent. The high sensitivity and speed of PCR suggest its potential routine implementation for diagnosing bovine mastitis caused by Streptococcus in any laboratory. Although, penicillin resistance was practically nonexistent, tetracycline and erythromycin exhibit higher resistance levels across all three species studied. In conclusion, the study underlines the importance of early diagnosis, highlights variations in bacterial prevalence, and proposes PCR as a valuable diagnostic tool for Streptococcus species responsible for bovine mastitis.

Risk factors and outcome of hepatitis C infection among patients in a secondary care hospital: A 5-year retrospective study.

BACKGROUND: Hepatitis C virus (HCV) infection, predominantly transmitted by exposure to infected blood, remains one of the major public health problems worldwide. This study aims to identify the risk factors of HCV transmission and its chronic complications among the study group. MATERIALS AND METHODS: This retrospective study was approved by the Research and Ethical Review and Approve Committee (RERAC) of Oman and conducted at a secondary-care hospital situated in the North Batinah region of Oman. The study population included all HCV cases confirmed by positive serology and reverse-transcription polymerase chain reaction tests during their presence at the hospital between January 2017 and December 2022. The relevant data of the study population were retrieved from the hospital electronic health record system. The data were analyzed using the Statistical Package for the Social Sciences (SPSS), Version 26.0. RESULTS: A total of 177 HCV confirmed cases were included in the study. HCV infection was predominant among males (74%) and individuals of the age group of 21-60 years (74.6%). Genotyping was possible only in 107 cases. Among HCV genotypes, genotype 3 (58.9%) was the most frequently identified, followed by genotype 1 (34.6%). Hemodialysis (21.5%), history of blood transfusion (16.4%), and injection drug use (11.9%) were the major risk factors for HCV infection, while cirrhosis (7.3%) and fatty liver disease (4%) were the most frequently observed chronic HCV complications. HCV infection in the spouse/partner (21.5%), alcohol use (7.3%), and co-infection with hepatitis B virus (2.3%) and human immunodeficiency virus (1.7%) were the other significant factors detected in our study population. CONCLUSIONS: HCV is a multi-factorial disease leading to severe chronic complications, thus representing a public health threat. This clearly emphasizes the cruciality of HCV community awareness campaigns and enhancement of Omani national guidelines for early screening of high-risk groups as well as effective management of HCV-infected cases to reduce the substantial burden of the disease on patients as well as the healthcare system.

Nitric Oxide Synthase 3 Gene Polymorphisms and Their Association with Acute Myocardial Infarction and Chronic Stable Angina: A Case-Control Study from Northern India.

Background: Coronary artery disease (CAD) that encompasses acute myocardial infarction (AMI), chronic stable angina (CSA), and unstable angina (UA) has numerous known risk factors. Genetic predispositions contribute as major risk in the development of CAD and the genes regulating atherosclerosis are important for disease prevention. Nitric oxide synthase 3 (NOS3) gene responsible for nitric oxide (NO) production is of special importance. Aim: To evaluate the role of three NOS3 polymorphisms (-786C/T, 894G/T, and 4a4b) in patients with CAD, particularly in AMI and CSA and their comparison with healthy controls. Materials and Methods: One hundred patients in each AMI and CSA group and 100 controls were included and were typed for three NOS3 polymorphisms (-786C/T, 894G/T, and 4a4b) by polymerase chain reaction-restriction fragment length polymorphism. Plasma NO metabolites (NOx) were also evaluated. Results: A significant association of 894G/T polymorphism with AMI in dominant model (P = 0.052) and with CSA in dominant and codominant models was detected (P = 0.008 and P = 0.006, respectively). Plasma NO levels were found to be significantly higher (P < 0.0001) in healthy controls (43.80 ± 6.28) compared to AMI and CSA patients (37.05 ± 6.75 and 38.67 ± 5.61). No significant association of -786C/T and 4a4b polymorphism with AMI and CSA risk under recessive, dominant, and codominant models was detected. Conclusion: Our study revealed a significant association of 894G/T polymorphism with AMI and independent association of NOx levels with CAD, indicating high risk of CAD in the North Indian population. Our findings will be helpful in identifying the genetic risk factors associated with CAD and better management of the diagnostic as well as therapeutic measures.

Identification and Validation of an Invasion-Related Disease-Free Survival Prognostic Model for Tongue Squamous Cell Carcinoma.

INTRODUCTION: Tongue squamous cell carcinoma (TSCC) is a common malignant tumour type with aggressive invasion and a poor prognosis. To date, invasion-related gene expression signatures for the prognostic stratification of TSCC patients are unavailable in clinical practice. This study aimed to assess the impact of invasion-related genes on the prognosis of TSCC patients. METHODS: We obtained mRNA profiles and clinical data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases (TCGA-TSCC and GSE41116, respectively). The TSCC samples from the TCGA-TSCC cohort were randomly divided into TCGA training and TCGA test datasets at a 7:3 ratio. Next, a disease-free survival (DFS) prognostic risk model was established on the basis of univariate and stepwise multivariate Cox regression analyses of the TCGA training cohort. Moreover, prognostic genes were screened. The model was subsequently evaluated and validated using the TCGA test and GSE41116 datasets. In addition, the prognostic genes were validated in the human TSCC cell line UM1 and the human oral keratinocyte (HOK) cell line using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: A total of 70 candidate genes related to invasion were identified in the TCGA-TSCC cohort. DFS data were subsequently constructed, and 6 prognostic genes, HMGN2, MYL12B, ACTB, PPP1CA, PSMB9, and IFITM3, were identified. The TSCC samples were divided into high- and low-risk groups in the TCGA training, TCGA test, and GSE41116 cohorts, respectively. In particular, patients with TSCC in the low-risk group had longer DFS than those in the high-risk group. Furthermore, qRT-PCR analysis confirmed that the expression levels of the 6 prognostic genes were significantly greater in the TSCC cell line UM1 than in the HOK cell line. CONCLUSION: This study identified new invasion-related target genes related to poor prognosis in TSCC patients, providing new insights into the underlying mechanisms of TSCC invasion.

Performance comparison of three commercial multiplex molecular panels for respiratory viruses at a South African academic hospital.

Background: Respiratory infections are a major contributor to hospital admissions. Identification of respiratory pathogens by means of conventional culture and serology methods remains challenging. Multiplex molecular assays are an appealing alternative that endeavours to be rapid, more accurate and less arduous. Objective: The study aimed to compare the clinical performance of three commercial multiplex molecular assays for respiratory viruses. Methods: Forty-eight respiratory specimens obtained from patients at Tygerberg Hospital in the Western Cape province of South Africa were studied. These specimens were collected between May 2020 and August 2020. The results of the Seegene Anyplex™ II RV16, FilmArray® Respiratory 2.1 plus Panel (FARP), and QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QRP) were analysed based on the overlapping targets. A composite reference standard was applied to provide a standard reference for comparison. Results: The overall sensitivity of the Seegene Anyplex™ II RV16 was 96.6% (57/59), the FARP 98.2% (56/57) and the QRP 80.7% (46/57). The overall specificities were 99.8% (660/661), 99.0% (704/711) and 99.7% (709/711), respectively. The QRP failed to detect coronaviruses and parainfluenza viruses in 41.7% (5/12) and 28.6% (4/14) of positive specimens, respectively, while the FARP produced the lowest target specificity of 88.4% (38/43) for rhinovirus/enterovirus. Conclusion: The overall specificity of all three platforms was comparable; however, the sensitivity of the QRP was inferior to that of the ARV and FARP. What this study adds: This study adds to the body of performance characteristics described for respiratory multiplex panels, especially in the African context where molecular diagnostics for infectious diseases are gaining momentum.

Impacts of circulating cytokine levels and gene polymorphism predisposition on type 1 diabetes mellitus.

PURPOSE: A wide range of cytokines has been demonstrated to be involved in the etiology of type 1 diabetes mellitus (T1DM). Gene polymorphisms may potentially contribute to a hereditary predisposition toward circulating cytokine levels as (high, intermediate, or low) since they can affect cytokine production or function. The aim of this study was to investigate the roles of cytokine levels and the association of single-nucleotide polymorphisms (SNPs) within cytokine genes with T1DM in Saudi children. METHODS: Totals of 91 well-characterized T1DM patients and 91 T1DM-free control subjects were enrolled in this study. RESULTS: The levels of 3 circulating cytokines (transforming growth factor [TGF]-ß1, interleukin [IL]-10, and IL-6) and 6 SNPs in 3 cytokine genes (TGF-ß1 [rs1800470 and rs1800471], IL-10 [rs1800896, rs1800871, and rs1800872], and IL-6 [rs1800795]) that contribute to genetic susceptibility were measured by enzyme-linked immunosorbent assay and polymerase chain reaction with sequence-specific primers. Our fn dings show that TGF-ß1 serum levels were signifcantly lower in the children with T1DM than in the control participants. The TGF-ß1 genotypes with a high-production phenotype were signifcantly less frequent and those with a lowproduction phenotype were signifcantly more frequent in the children with T1DM compared to the control participants. respectively. Furthermore, the IL-6 genotype frequency with low level of IL-6 production were signifcantly increased in the T1DM group compared to the control group. Moreover, our data demonstrated no appreciable diferences in circulating serum level or genotype and phenotype of IL- 10 between the patients and controls. CONCLUSION: This kind of measurement, which considers the prediction of T1DM, may be useful in assessing the severity of T1DM and susceptibility to T1DM among Saudi children.

Assessing knowledge and awareness levels regarding cervical cancer and HPV vaccination in the Jammu regions.

Background and Objectives: Cervical cancer global burden is highly skewed towards poor countries primarily due to lack of awareness, poor screening, and low uptake of prophylactic vaccines. The purpose of our study is to educate and raise awareness among young girls and women about the importance of cervical screening and HPV vaccination. Materials and Methods: The present study, conducted from January 2023 to December 2023, focused on students, teachers, housewives, and healthcare professionals in the Jammu region to assess their awareness of cervical cancer and the HPV vaccine. HPV DNA testing was carried out using the Truenat Real-Time PCR method at Swastik Diagnostic Laboratory, Jammu. Results: Knowledge of cervical cancer, awareness of the HPV virus, and the vaccination status of women were assessed in survey. In the HPV screening test, out of 2,400 women, 106 tested positive for HPV. Among these 106 women, 19% had a high viral load (Ct < 20), 11% had a low viral load (25 ≤ Ct < 30), indicating a low relative concentration of HPV viruses, 40% had a medium viral load (20 ≤ Ct < 25), and 30% had very low viral loads (Ct ≥ 30). Conclusion: These findings highlight the importance of routine cervical screenings, such as Pap smears and HPV tests, for the early detection of cervical cancer. There is an urgent need to implement cervical cancer screening and vaccination programs in the Jammu region.

Diagnostic value of antibody testing in comparison with lung scan and PCR in patients suspected of having COVID-19.

Background and Objectives: SARS-CoV-2 is a newly discovered viral infection. It's still unclear how antibodies react in infected individuals, and there is not enough evidence to support the clinical use of antibody examination. This study evaluates the diagnostic value of serologic tests for diagnosing COVID-19. Materials and Methods: 32 patients for whom serologic testing was performed within 7 to 21 days from symptom onset and whether they were diagnosed with COVID-19 by both PCR and lung HRCT as gold standard tests at the same time, were included in the study. Results: Serologic tests (IgM / IgG) compared to PCR and lung HRCT scan to diagnose COVID-19, were 89.3% specific and 59.6% sensitive. Positive predictive value (PPV) was 95% and negative predictive value (NPV) was 37%. The diagnostic accuracy index of the serologic test was 0.745 (CI 0.651-0.838) (p-value <0.001). Conclusion: Serologic testing can be a complementary alternative for SARA-CoV-2 nucleic acid RT-PCR, although it cannot replace it completely. IgG/IgM combo test kits and RT-PCR together can give more insight into the diagnosis of SARS-CoV-2.

Relationship Between IL-10 Single Nucleotide Polymorphisms (rs1800871, rs1800872, and rs1800896) and the Severity of COVID-19.

Background: Interleukin-10 (IL-10) is an anti-inflammatory cytokine whose levels are elevated in patients with severe COVID-19. IL-10 polymorphisms may play a role in increasing IL-10 levels and the severity of COVID-19. This study aimed to investigate the relationship between IL-10 single nucleotide polymorphisms (SNPs) (rs1800896 [-1082 C < T], rs1800871 [-819 A > G], and rs1800872 [-592 T > G]) and the severity of COVID-19 in patients from Kermanshah Province, Iran. Methods: A total of 150 patients with mild COVID-19 (84 men and 66 women aged 40.1 ± 12.44 years) and 143 patients with severe COVID-19 (76 men and 67 women aged 61.04 ± 15.65 years) participated in this study. Blood samples were collected from the patients, DNA was extracted, and the genotype of each SNPs was determined using the polymerase chain reaction-restriction fragment length polymorphism method. Result: The results of this study did not show a significant relationship between the genotypes of the three studied SNPs and the severity of COVID-19 (p > 0.05). Conclusion: According to our findings, these SNPs were not associated with COVID-19 severity in patients in Kermanshah.

Association between real-time polymerase chain reaction cycle threshold value and clinical presentation in children with Bordetella pertussis.

BACKGROUND: The cycle threshold (Ct) value is inversely proportional to the number of copies of the target region in a sample, suggesting that a low Ct value indicates a high pathogen load. The relationship between Ct value and clinical presentation in children with pertussis is not well-defined. METHODS: We investigated the relationships between the Ct value of nasopharyngeal samples positive for Bordetella pertussis deoxyribonucleic acid via real-time polymerase chain reaction (PCR), collected from children on admission and their adult family members between May 2022 and March 2024 at Hangzhou Children's Hospital, China. The study focused on the correlation between Ct value and clinical presentation in children with pertussis. RESULTS: The Ct value was positively correlated with age (r = 0.362, P = 0.001). The mean Ct value for children with pertussis was 28.0 (range: 22.0-32.0), which was lower than the 32.0 (range: 30.0-34.0) observed in adults. Ct value was inversely correlated with length of stay, an indicator of disease severity (r = -0.356, P = 0.001). Logistic regression analyses revealed that both Ct value (OR: 0.891, 95% CI: 0.799-0.993, P = 0.036) and white blood cell count (OR: 1.127, 95% CI: 1.005-1.263, P = 0.040) were independently associated with severity of pertussis. CONCLUSIONS: Real-time PCR Ct values at initial diagnosis for pertussis may potentially predict severe disease outcomes in children.

New method to induce neurotrophin gene expression in human adipose-derived stem cells in vitro.

Rosemary leaf extract, a well-known medicinal plant, can induce neurotrophin gene expression and proliferation in stem cells. Human adipose-derived stem cells (hASCs) with high proliferation and differentiation capacity are easily accessible and can be extracted with the least damage. This study evaluated the effect of rosemary extract (RE) on neurotrophin gene expression at 48 h postinduction in hASCs. hASCs were isolated from healthy female donors, aged 28-35 years, who had undergone abdominal liposuction. Passage-4 stem cells were cultured and treated with different doses of RE (from 30 to 70 µg/ml) containing 40% carnosic acid for 48 h. Reverse transcription-polymerase chain reaction was used to check the expression of neurotrophin genes. The expression of NTF3, NTF4, and nerve growth factor genes in cells treated with 40-60 µg/ml and the expression of GDNF in cells treated with 50-70 µg/ml of RE for 48 h showed a significant increase compared to cells cultured in serum-containing medium. However, different doses of RE showed no effect on brain-derived neurotrophic factor gene expression in the treated cells. RE (50, 60 µg/ml) leads to an increase of neurotrophin gene expression in hASCs as compared to routine cell culture. Hence, this protocol can be used to prepare ideal cell sources for cell therapy.

DEVELOPMENT OF A RAPID SNP PCR ASSAY TO DIFFERENTIATE BETWEEN EUROPEAN AND NORTH AMERICAN HAPLOTYPES OF ECHINOCOCCUS MULTILOCULARIS.

Herein we describe a single nucleotide polymorphism-specific polymerase chain reaction (PCR) assay to rapidly detect and differentiate variants belonging to the European and North American lineages of Echinococcus multilocularis in clinical samples. This is an extremely relevant and applicable test in North America because the range of E. multilocularis continues to expand across the continent and because of a rise in prevalence in wildlife, domestic animals, and humans. The endemic North American (NA) and introduced European (EU) variants are believed to have different pathogenic potentials, with the EU variants being more infective and pathogenic than the NA variants. The rise of the EU variants of E. multilocularis increases the risk of spillover from wildlife to humans because of its increased potential for infectivity. Current PCR-based diagnostics can detect E. multilocularis deoxyribonucleic acid (DNA), but DNA sequencing is required to identify the specific variant. Our assay provides a straightforward conventional PCR method to differentiate the NA and EU variants, and we suggest this same approach could be used for the diagnosis of other parasites or variants that are genetically very similar. As surveillance continues for E. multilocularis across North America, identifying the different genetic variants from different geographic regions will become essential to understanding the current epidemiological shift that the parasite is experiencing, as well as informing public health decisions in affected areas.

Knee tuberculosis: an overlooked clinical entity.

INTRODUCTION: The most common anatomic sites affected by extrapulmonary tuberculosis are lymph nodes, pleura, bones, and joints, urogenital tract, and meninges. Tuberculous arthritis is difficult to diagnose early because of its atypical insidious clinical manifestations and non-specific imaging findings. CASE REPORT: A 59-year-old male presented with progressive swelling in his left knee for over two months. The patient was initially misdiagnosed with pigmented villonodular synovitis (PVNS) and had undergone total knee arthroplasty (TKA) two years ago, however, the TKA did not completely alleviate his symptoms. Comprehensive radiological and laboratory assessments, including X-rays, magnetic resonance imaging and computed tomography scans, and an interferon-γ release assay (IGRA), pointed towards a diagnosis of tuberculous knee arthritis. Definitive diagnosis was established through the detection of Mycobacterium tuberculosis (MTB) DNA in the synovial fluid via polymerase chain reaction (PCR) and a positive IGRA result. CONCLUSIONS: The case underscores the importance of considering MTB infection in the differential diagnosis of chronic unilateral knee arthritis, especially given the atypical clinical manifestations and imaging findings that can mimic other conditions like PVNS.

Diagnostic Accuracy of Dermatoscopy Versus Microbiological Culture and Polymerase Chain Reaction in the Diagnosis of Onychomycosis: A Cross-Sectional Study.

BACKGROUND: Several clinical signs in dermatoscopy are very characteristic of onychomycosis and can be a quick complement for the diagnosis of onychomycosis. OBJECTIVES: The aim of this study was to evaluate the diagnostic accuracy of dermatoscopy compared to microbiological culture and polymerase chain reaction (PCR), as well as the clinical signs associated with onychomycosis. METHODS: The clinical signs of 125 patients were assessed cross-sectionally using dermatoscopy, and a positive or negative result was assigned. A sample was then taken for PCR and microbiological culture. RESULTS: Of the 125 patients, 69.6% (87/125) had positive results when both laboratory tests were combined. When they were not combined, the prevalence was lower at 48% (60/125) with PCR and at 43.2% (54/125) with culture. Furthermore, 76.8% (96/125) were classified as positive with dermatoscopy with a sensitivity of 1, a specificity of 0.76, positive predictive value of 0.91 and negative predictive value of 1 (with 95% confidence intervals). Of the 96 dermatoscopy-positive samples, 36 were negative with PCR (p < 0.001), 42 were negative with culture (p < 0.001) and nine were negative when both tests were combined (p < 0.001). Clinical signs that were significantly associated with the presence of onychomycosis were subungual hyperkeratosis (dermatoscopy: p = 0.004, odds ratio (OR) = 2.438; PCR + microbiological culture: p = 0.004, OR = 3.221), subungual detritus (p = 0.033, OR = 3.01, only with dermatoscopy) and dermatophytoma (dermatoscopy: p = 0.049, OR = 3.02; PCR + microbiological culture: p = 0.022, OR = 2.40). CONCLUSIONS: The results suggest that dermatoscopy is a good tool for the diagnosis of onychomycosis but should be used as a complementary test or for screening patients to be sampled for laboratory testing. The combination of the three tests can lead to a reduction of false-positive and false-negative clinical and laboratory results. This allows for early diagnosis and specific treatment based on test results.