Specific Polymerase Chain Reaction Research Articles

A Comparative Study of the Rate of Helicobacter pylori infection Using Three Diagnostic Techniques among Patients in Rivers State

Background: Approximately 89% of gastric cancers are attributable to H. pylori infection, however, only 1 in 5 patients survive longer than 5 years after diagnosis. Evidence has shown that screening and eradication of H. pylori in young adults would be cost-effective and could prevent 1 gastric cancer in every 4 to 6 cases. Diagnostic tests used to detect Helicobacter pylori are either invasive or noninvasive methods. These tests have varying sensitivity and specificity, having about 90% accuracy in the diagnosis of H. pylori infection in clinical practice. Aim: This study aims at detecting Helicobacter pylori using antibody-antigen, stool antigen and polymerase chain reaction (PCR) and to compare the infection rate among the different diagnostic techniques. Methodology: This was a cross-sectional study. The participants included attendants to tertiary and private hospitals in Port Harcourt metropolis for medical treatment. Blood and stool samples were collected from one hundred and eighty (180) subjects aged 1 to 60 years and above. Ethical approval for the study was obtained from the Rivers State University Teaching Hospital Ethics Committee. Convenience sampling technique was deployed. Samples were collected from subjects who consented to the study. All samples collected were analyzed in Medical Microbiology Laboratory, Rivers State Teaching Hospital and subsequently, molecular analysis was carried out. Results: The blood antibodies test (serology) method shows a greater number of positive cases (43.9%) than stool antigen method (8.3%). Also, out of the 75% stool antigen positive samples, subjected to more specific PCR, only 25% were positive. There was a significant difference (p-value of <0.001) in sensitivity between antigen and serology, there was also a significant difference (p-value of <0.025) between antigen and PCR result outcomes. Conclusion: The study has shown that there is a notable variation in the diagnosis of H pylori among the laboratory techniques used.

Immunohistochemical Analyses of Monkeypox Skin Lesions with MPXV A29 and A35 Antibodies: A Novel Insight for Clinical-Histopathological Forms.

The aim of this study was to describe the clinical, histopathological, and immunohistochemical characteristics of MPX and offer meaningful insights into the clinicopathology of MPX. We recruited eight men who had sex with men diagnosed with MPX based on positive results from MPX Virus (MPXV)-specific polymerase chain reaction. Skin biopsies were obtained from four selected lesions, including typical and atypical lesions. Histopathological examinations of atypical solitary ulceration revealed infiltrating inflammatory cells predominantly composed of plasma cells and lymphocytes, forming a "sleeve" around the superficial vessels of the dermis. These features might be misinterpreted as indicative of cutaneous syphilis infection. Meanwhile, typical pustular lesions displayed central necrotic epidermis accompanied by perivascular inflammatory infiltrate dominated by neutrophils, as well as ballooning and reticular degeneration of keratinocytes. Additionally, multinucleated keratinocytes and eosinophilic cytoplasmic inclusions known as Guarnieri's bodies were also observed. Importantly, this study represented the pioneering report on immunohistochemical detection of MPXV A29 and A35 proteins in skin lesions, distinguishing it from previous studies that focused on detecting vaccinia virus protein. The anti-MPXV A29 antibody exhibited robust cytoplasmic staining specifically within affected keratinocytes in both adjacent epidermis and hair follicles, thereby it could contribute to the diagnosis of MPX, especially for cases with atypical skin lesions.

THE PREVALENCE OF (OMPA, CSUE) GENES AMONG BIOFILM PRODUCER ACINETOBACTER BAUMANNII ISOLATES

Acinetobacter baumannii is a significant public health problem because it is capable of forming biofilms that may be responsible for the survival of this pathogen in the hospital environment. The aim of this study is to determine the role of (OmpA, CsuE) genes in biofilm production among A. baumannii isolates. A total of 100 clinical isolates of bacteria were collected from a different sources. All isolates were identified by biochemical test, specific selective media (CHROMagar) and polymerase chain reaction by detection presence of (blaOXA51) gene. only 60 isolates were diagnosed as A. baumannii, and to determine the biofilm production capacity two different methods were used Congo red agar method(CRA) and Microtiter plate method (MTP). MTP method result showed 85% of isolates were able to form biofilms, on the other hand, 33.33% of isolates had a biofilm production ability by showing bright black colonies on CRA. Conventional PCR was used to detect the presence of (OmpA, CsuE) genes and the result showed the prevalence of both genes in all isolates were 97%(58/60). It also shows a strong correlation between the presence of these genes and biofilm formation.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Comparative evaluation of MIC values of Trichosporon spp. by MTT assay and CLSI M27-A3 broth microdilution reference methods

Objectives: The objective of this study was to determine and compare the minimum inhibitory concentration (MIC) values of Trichosporon spp. by MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl- 2H-tetrazoliumbromide) assay, and Clinical and Laboratory Standards Institute M27-3rd edition (CLSI M27-A3) broth microdilution methods. Materials and Methods: Antifungal susceptibility testing was done by CLSI M27-A3 broth microdilution and MTT assay for all the 72 Trichosporon isolates after genus specific and Trichosporon asahii specific polymerase chain reaction (PCR). Candida krusei ATCC 6258 was used as the reference strain. Statistical Analysis: All statistical data were analyzed using the Statistical Package for the Social Sciences, version 17 for Microsoft Windows. The percentage of agreement was calculated using the Type C intraclass correlation coefficient. Results: The MICs by MTT assay strongly correlated with those obtained by CLSI M27-A3 method, by being either the same or within 1 dilution of MIC by CLSI method. Furthermore, the ranges of MICs obtained by MTT and CLSI method were all identical in our study. The overall agreement between the two methods for the Trichosporon isolates was good, that is, 90.8% in our study. Conclusions: MTT assay can be an alternative method that assists reading of MICs visually with a colored end point, making it easier compared to CLSI M27-A3 method. MTT assay can also be standardized for other yeasts and molds so that antifungal susceptibility tests can be done for different fungi.

Nested PCR Targeting the Flagellin Gene (fliC) in Urine Sample for Diagnosis of Typhoid Fever – An Internal Pilot Study

Abstract Introduction Typhoid fever remains a significant public health challenge, especially in developing countries. Diagnostic complexities arise from the disease’s non-specific clinical manifestations that overlap with other febrile illnesses. Hence, reliable laboratory testing is crucial. Nested polymerase chain reaction (PCR) stands out for its diagnostic performance, especially when utilizing urine due to its minimal PCR inhibitors and ease of collection. Aim The study aimed to test the performance of Salmonella enterica serotype Typhi (Salmonella Typhi) fliC gene specific nested PCR in urine for laboratory diagnosis of typhoid fever. Subjects and Methods This observational, cross-sectional study was conducted as an internal pilot study during the period from August 2021 to October 2022. It included 25 clinically diagnosed enteric fever patients, recruited from Al-Abasseya Fever Hospital, Cairo, Egypt, and 25 healthy individuals as controls, matched for age and sex. Nested PCR was performed to detect the fliC gene of Salmonella Typhi in urine samples from both patients and controls. Urine culture was conducted for subjects in both groups, while blood and stool cultures were exclusively performed for the patients' group. Results The sensitivity of the nested PCR test targeting the fliC gene in urine was 44%, with a specificity of 88%. The positive predictive value was 78.57%, the negative predictive value was 61.11%, the positive likelihood ratio was 3.7, and the negative likelihood ratio was 0.63. Blood culture isolation exhibited a very low positivity rate of 4%. Conclusion Nested PCR targeting the fliC gene in urine samples holds promise as a diagnostic tool for typhoid fever. Despite its moderate sensitivity, the relatively higher specificity suggests its potential utility in confirming typhoid fever cases. The test offers a valuable adjunctive tool alongside conventional diagnostic method. However, further research and validation may be necessary before widespread clinical application. The performance of the test needs to be evaluated in clinically diagnosed and laboratory confirmed cases of typhoid fever.

Identification of activating somatic mutations in the <i>PIK3CA</i> gene in breast tumors and determination of their minimal set for clinical diagnostic testing

Background: For effective screening of breast cancer patients for candidates for target therapy with alpelisib, it is necessary to identify activating somatic mutations in the PIK3CA gene by allele specific polymerase chain reaction (PCR); this requires that an optimal list of mutations should be compiled. Aim: To determine the spectrum of somatic mutations in the PIK3CA gene in breast cancer tumors by means of high performance sequencing (next generation sequencing, NGS) and to identify their minimal set for clinical diagnostic testing by allele specific PCR. Methods: Targeted NGS was used to identify mutations in the PIK3CA gene in DNA obtained from paraffin blocks with tumor material from 431 patients with HR+HER2- breast cancer. A set of the most common somatic mutations was also detected by allele specific PCR. Results: We have developed a set of reagents and a protocol for targeted NGS of frequently mutating regions of the PIK3CA and ESR1 genes, which was used to analyze samples from 451 HR+/HER2- breast cancer patients. Clinically significant activating mutations in the PIK3CA gene were found in 32.7% of the samples (141/431). The frequency of the mutant allele ranged from 0.15 to 0.65. Six mutations were most common: c.3140AG p.His1047Arg (69), c.1633GA p.Glu545Lys (32), c.1035TA p.Asn345Lys (12), c.1624GA p.Glu542Lys (9), c.3140AT p.His1047Leu (8), Cys420Arg c.1258TC (3). In total, these mutations amounted to 94.3% (133/141). In 3.5% of the samples (15/431), there were clinically significant somatic mutations in the ESR1 gene: c.1613AG p, Asp538Gly (7), c.1610AC p.Tyr537Ser (6), c.1609TA p.Tyr537Asn (1), c.1610AG p.Tyr537Cys (1), causing resistance to hormone therapy in patients with breast cancer. While rare mutations comprised only 5.7% of our sample, we validated a set of reagents to identify the six mutations described above by allele specific PCR. NGS and PCR were completely concordant. Conclusion: PCR testing of activating somatic mutations of the PIK3CA gene meets the requirements for sensitivity ( 90%) and specificity (100%) for a clinical test and can be used in the selection of patients for targeted therapy with PIK3CA inhibitors.

Biosensing platforms for DNA diagnostics based on CRISPR/Cas nucleases: towards the detection of nucleic acids at the level of single molecules in non-laboratory settings.

The use of CRISPR/Cas nucleases for the development of DNA diagnostic systems in out-of-laboratory conditions (point-of-need testing, PONT) has demonstrated rapid growth in the last few years, starting with the appearance in 2017-2018 of the first diagnostic platforms known as DETECTR and SHERLOCK. The platforms are based on a combination of methods of nucleic acid isothermal amplification with selective CRISPR/Cas detection of target amplicons. This significantly improves the sensitivity and specificity of PONT, making them comparable with or even superior to the sensitivity and specificity of polymerase chain reaction, considered as the "gold standard" of DNA diagnostics. The review considers modern approaches to the coupling of CRISPR/Cas detection using Cas9, Cas12a, Cas12b, Cas13a, Cas14, and Cas3 nucleases to various methods of nucleic acid isothermal amplification, with an emphasis on works in which sensitivity at the level of single molecules (attomolar and subattomolar concentrations of the target) is achieved. The properties of CRISPR/Cas nucleases used for targeted DNA diagnostics and the features of methods of nucleic acid isothermal amplification are briefly considered in the context of the development of diagnostic biosensing platforms. Special attention is paid to the most promising directions for the development of DNA diagnostics using CRISPR/Cas nuclease.

A comprehensive investigation into the ranges of laboratory tests present in cerebrospinal fluid across various types of meningitis within different age categories

Background. Diagnosing meningitis requires a lumbar puncture to analyze cerebrospinal fluid (CSF) and determine the causative pathogen. Key diagnostic measures include Glucose and Protein concentrations, cell count, differential leukocyte count, Gram stain, culture, and PCR if necessary. Objective. Our analysis assessed patterns in key biomarkers—neutrophils, lymphocytes, cerebrospinal fluid white blood cell count, and CSF/blood Glucose and Protein ratios—across various types of meningitis, including meningococcemia, meningococcal, tuberculous, pneumococcal, aseptic, and other bacterial meningitis. We examined these biomarkers within distinct age categories: children (0-12 years), adults (13-64 years), and the elderly (≥ 65 years). Methods. Descriptive statistics were used to analyze various types of meningitis across different age groups. We assessed the normality of laboratory biomarkers using the Shapiro-Wilk test, and applied Welch ANOVA to explore potential differences in biomarker levels among various types of meningitis. A Games-Howell post hoc test was used to identify specific pairs of meningitis types with statistically significant differences in biomarker levels. Results. The analysis of variance of cerebrospinal fluid biomarkers revealed significant differences in CSF WCC (cells/mm3), Neutrophils (%), Lymphocytes (%), CSF/blood Glucose and Protein ratios in children and adults (p <0.05). However, differences were observed solely in neutrophil levels among elderly subjects. In bacterial meningitis, neutrophil levels increased across all ages, with adults showing higher levels. For meningococcal meningitis, adults had a median neutrophil level of 78.5% [60%, 90%] compared to 57% [24.5%, 85.25%] in children. The CSF white cell count (WCC) median was also higher in adults (339 cells/mm³) compared to children (224 cells/mm³). Viral meningitis cases exhibited higher lymphocyte levels across all age groups, with medians of 69% [34%, 87%] in children, 82% [61%, 93%] in adults, and 77% [55%, 90%] in the elderly. The glucose ratio was lower in bacterial meningitis (<0.3) and higher in viral cases (>0.5). Protein ratios were elevated in bacterial meningitis, indicating increased blood-brain barrier permeability. These results demonstrate a distinct biological profile for different causative agents, modulated by patient age.

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Epidemiology of travel-associated dengue from 2007 to 2022: A GeoSentinel analysis.

Dengue is a leading cause of febrile illness among international travellers. We aimed to describe the epidemiology and clinical characteristics of imported dengue in returning travellers evaluated at GeoSentinel sites from 2007 to 2022. We retrieved GeoSentinel records of dengue among travellers residing in non-endemic countries. We considered dengue confirmed when diagnosed by a positive dengue virus (DENV)-specific reverse-transcriptase polymerase chain reaction, positive NS-1 antigen and/or anti-DENV IgG seroconversion, and probable when diagnosed by single anti-DENV IgM or high-titre anti-DENV IgG detection. Severe dengue was defined as evidence of clinically significant plasma leakage or bleeding, organ failure, or shock, according to the 2009 World Health Organization guidance. Complicated dengue was defined as either severe dengue or dengue with presence of any warning sign. Analyses were descriptive. This analysis included 5958 travellers with confirmed (n = 4859; 81.6%) or probable (n = 1099; 18.4%) dengue. The median age was 33 years (range: <1-91); 3007 (50.5%) travellers were female. The median travel duration was 21 days (interquartile range [IQR]: 15-32). The median time between illness onset and GeoSentinel site visit was 7 days (IQR: 4-15). The most frequent reasons for travel were tourism (67.3%), visiting friends or relatives (12.2%) and business (11.0%). The most frequent regions of acquisition were South East Asia (50.4%), South Central Asia (14.9%), the Caribbean (10.9%) and South America (9.2%). Ninety-five (1.6%) travellers had complicated dengue, of whom 27 (0.5%) had severe dengue and one died. Of 2710 travellers with data available, 724 (26.7%) were hospitalized. The largest number of cases (n = 835) was reported in 2019. A broad range of international travellers should be aware of the risk of acquiring dengue and receive appropriate pre-travel counselling regarding preventive measures. Prospective cohort studies are needed to further elucidate dengue risk by destination and over time, as well as severe outcomes and prolonged morbidity (long dengue) due to travel-related dengue.

Discovery and characterization of novel jeilongviruses in wild rodents from Hubei, China

The genus Jeilongvirus comprises non-segmented negative-stranded RNA viruses that are classified within the Paramyxoviridae family by phylogeny. Jeilongviruses are found in various reservoirs, including rodents and bats. Rodents are typical viral reservoirs with diverse spectra and zoonotic potential. Little is currently known about jeilongviruses in rodents from central China. The study utilized high-throughput and Sanger sequencing to obtain jeilongvirus genomes, including those of two novel strains (HBJZ120/CHN/2021 (17,468 nt) and HBJZ157/CHN/2021 (19,143 nt)) and three known viruses (HBXN18/CHN/2021 (19,212 nt), HBJZ10/CHN/2021 (19,700 nt), HBJM106/CHN/2021 (18,871 nt)), which were characterized by genome structure, identity matrix, and phylogenetic analysis. Jeilongviruses were classified into three subclades based on their topology, phylogeny, and hosts. Based on the amino acid sequence identities and phylogenetic analysis of the L protein, HBJZ120/CHN/2021 and HBJZ157/CHN/2021 were found to be strains rather than novel species. Additionally, according to specific polymerase chain reaction screening, the positive percentage of Beilong virus in Hubei was 6.38%, suggesting that Beilong virus, belonging to the Jeilongvirus genus, is likely to be widespread in wild rodents. The identification of novel strains further elucidated the genomic diversity of jeilongviruses. Additionally, the prevalence of jeilongviruses in Hubei, China, was profiled, establishing a foundation for the surveillance and early warning of emerging paramyxoviruses.

Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility.

Due to its decade-long progression, colorectal cancer (CRC) is most suitable for population screening to achieve a significant reduction in its incidence and mortality. DNA methylation has emerged as a potential marker for the early detection of CRC. However, the current mainstream methylation detection method represented by bisulfite conversion has issues such as tedious operation, DNA damage, and unsatisfactory sensitivity. Herein, a new high-performance CRC screening tool based on the promising specific terminal-mediated polymerase chain reaction (STEM-PCR) strategy is developed. CRC-related methylation-specific candidate CpG sites are first prescreened through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases using self-developed bioinformatics. Next, 9 homebrew colorectal cancer DNA methylated STEM‒PCR assays (ColoC-mSTEM) with high sensitivity (0.1%) and high specificity are established to identify candidate sites. The clinical diagnostic performance of these selected methylation sites is confirmed and validated by a case-control study. The optimized diagnostic model has an overall sensitivity of 94.8% and a specificity of 95.0% for detecting early-stage CRC. Taken together, ColoC-mSTEM, based on a single methylation-specific site, is a promising diagnostic approach for the early detection of CRC which is perfectly suitable for the screening needs of CRC in primary healthcare institutions.

#2436 Genetic variability in rosuvastatin metabolism explains rhabdomyolysis and consequent acute renal failure

Abstract Background and Aims Low density lipoprotein cholesterol is an independent risk factor for atherosclerotic cardiovascular disease. Rosuvastatin is one of the most frequently prescribed statins and also one of the most frequent causes of rhabdomyolysis with consequent acute renal failure (ARF). Risk factors for statin induced rhabdomyolysis, i.e. the type of statin and its dose, patient's age, kidney and liver function, gender and concurrent treatment with cytochrome P450 (CYP) inhibitors, should be considered at the time of statin prescription. Single nucleotide polymorphisms (SNPs) in genes coding the transport proteins (SLCO1B1, ABCG2) and CYP2C9, catalysing rosuvastatin oxidation, may lead to decreased protein function leading to accumulation of plasma rosuvastatin which increases the risk of rhabdomyolysis. Herewith we present a case of a female patient admitted to our department due to rhabdomyolysis and ARF with the aim of alerting against uncritical high dose rosuvastatin prescribing and of pointing out the role of genetic variability in drug metabolizing enzymes and transporters (DMET) in rosuvastatin side effects. Method a 79-years old Caucasian woman admitted to our department due to rhabdomyolysis and ARF was genotyped for polymorphisms in DMET genes coding for CYP2C9 (rs1799853, rs1057910, rs28371686, rs28371685), SLCO1B1 (rs4149056, rs2306283) and ABCG2 (rs2231142) using competitive allele specific polymerase chain reaction (KASPar). Results Presented patient suffered from an acute myocardial infarction in October 2023. She was treated with percutaneous coronary intervention, antiaggregation therapy and hypolipidemic therapy (rosuvastatin 40 mg/ezetimibe 10 mg). Her creatinine was 55 μM (eGFR 61 ml/min) at the time of statin prescription. Several months later she was admitted septic with myoglobin &amp;gt;50.000 μg/L and creatinine approximately 1600 μM. Infection was treated with piperacillin/tazobactam. Myoglobin was removed with Theranova dialyzer. The patient was dismissed home two weeks later with creatinine 230 μM. As potential patient's risk factors for rhabdomyolysis we considered high dose of rosuvastatin, patient's higher age and slower rosuvastatin disposition due to SNPs in DMET. Sepsis may have been an additional contributory factor. Genotyping revealed polymorphisms in all three genes involved in rosuvastatin metabolism and transport: CYP2C9, SLCO1B1 and ABCG2 (Table 1). Two genotypes resulted in the phenotypes with slower rosuvastatin metabolism, probably leading to gradual accumulation of plasma rosuvastatin, which may have manifested in rhabdomyolysis that contributed to ARF. Heterozygous CYP2C9*2 and *3 carriers have, in comparison to the wild type carriers, approximately 30% and 80% slower rosuvastatin metabolism, respectively. Heterozygous ABCG2 c.421C&amp;gt;A carrier state may also contribute slightly to rosuvastatin accumulation. Furthermore, the potential interaction with inflammation should not be overlooked, as inflammation may have a major effect on drug metabolism and transport through downregulation of CYP enzymes as well as drug transporters. If genotypes of the respective DMET genes were known in advance, high dose rosuvastatin could have been avoided despite relatively good kidney function at the time of rosuvastatin prescription. Conclusion Statins should be prescribed with caution and their dose adapted to the risk factors that should always be checked for at the time of statin prescription. We recommend periodical surveillance of serum creatinine, eGFR, urine protein to creatinine ratio, liver tests, creatine kinase and myoglobin in patients treated with high dose statins. Furthermore, SLCO1B1, ABCG2 and CYP2C9 genotyping before rosuvastatin prescription would enable insight into its metabolism. Genotype information could lead to more cautious statin prescribing, especially in the presence of other factors, such as infection or concomitant drug treatment that may modify the activity of DMET. In the case of chronic kidney disease drug dosing should be appropriately adjusted in accordance with drug's Summary of product characteristics and KDIGO guidelines.

A putatively novel papillomavirus associated with cutaneous plaques and squamous cell carcinoma in captive North American snow leopards (Panthera uncia).

Cutaneous plaques and squamous cell carcinoma (SCC) are common in captive North American snow leopards (SLs) (Panthera uncia). Our objective was to determine whether these lesions are potentially associated with papillomavirus(es). Polymerase chain reaction (PCR) was performed on 3 cutaneous plaques using degenerate primers for papillomaviruses. A putatively novel papillomavirus was identified that shared 76% sequence identity to Felis catus papillomavirus 2. Specific PCR for this virus was performed on 5 cutaneous SCC samples and 7 normal skin samples, which were all positive. In situ hybridization for this putatively novel virus was performed, which revealed strong hybridization signals within hyperplastic cells in cutaneous plaques (n = 3) and within neoplastic cells in cutaneous SCC samples (n = 5). No hybridization signals were identified within normal skin. Ultimately, identification of a causal viral agent in the development of plaques and SCC in SLs will help guide therapeutic intervention and lay the foundation for development of prophylactic vaccines.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Prognostic value of BRCA1 promoter methylation for patients with epithelial ovarian cancer

ObjectiveBRCA1 promoter methylation (BRCA1pm) is suspected to alter prognosis of patients with epithelial ovarian cancer (EOC). We aimed to evaluate the prognostic impact of this epigenetic modification. MethodsWe conducted a retrospective, monocentric study from 11/2006 to 08/2018. Patients with EOC and available status concerning somatic BRCA1/2 mutation and BRCA1pm were included. Three groups were defined: patients without BRCA1/2 mutation or BRCA1pm, patients with BRCA1/2 mutation and patients with BRCA1pm. BRCA1/2 mutations were analyzed in current care settings by next-generation sequencing (NGS). BRCA1pm analysis was assessed and quantified from bisulfite converted DNAs using fluorescent methylation specific polymerase chain reaction (PCR) and fragment analysis. All patients signed a consent form and the study was authorized by a Personal Protection Committee. Descriptive statistics were used to describe groups. Multivariate analysis was performed using the logistic regression model and including the variables that could be known at the time of diagnosis and that were significant at univariate analysis. Survival was compared between the groups. Kaplan-Mayer curves were used to express the differences in survival that were compared using log rank tests. Results145 patients were included: 95 (65.5 %) patients without BRCA1/2 mutation or BRCA1pm, 32 (22.1 %) patients with BRCA1/2 mutation, 18 (12.4 %) patients with BRCA1pm. Median survival was decreased in patients with BRCA1pm. Comparison of survival revealed a significant difference in overall survival (p = 0.0078) with a worse prognosis for patients with a BRCA1pm. ConclusionBRCA1pm in patients with EOC is an independent factor associated with a decreased overall survival. SynopsisBRCA1 promotor methylation in patients with epithelial ovarian cancer is an independent factor associated with a decreased overall survival.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

Role of ABCC2 gene polymorphism on deferasirox hepatic toxicity in thalassemia patients

Objectives: To investigate the relationship between ABCC2 gene polymorphism (G&gt;A, rs 8187710) and (C&gt;T, rs 717620) and hepatotoxicity in thalassemia patient treated deferasirox . Methods: The present work included a cross sectional study, 100 patients suffering iron overload and thalassemia out of 650 patients in (thalassemia center) and 100 healthy patients to compare the parameters of liver function tests of thalassemia patients with healthy group. The allele specific polymerase chain reaction technique (pcr) was used to detect the (G&gt;A, rs 8187710) and (C&gt;T, rs 717620 single nucleotide polymorphism (SNP). Results : 58 patients with rs 8187710 who are wild (GG) and 32 patients hetero(GA)and 56 patients with rs 717620 who wild (CC) and 31 hetero (CT) are exposed to hepatotoxicity than patients with homo groups Conclusion: According to the results of the currents study, the (G&gt;A, rs 8187710) and (C&gt;T, rs 717620) single nucleotide polymorphism (SNP) was associated with hepatotoxicity in patients treated deferasirox.

Molecular Analysis of Antibiotic Resistance in CarbapenemResistant Pseudomonas aeruginosa Isolates Isolated from Clinical Samples

Pseudomonas aeruginosa is an opportunistic pathogen that causes increased morbidity and mortality in risky patient groups. Nowadays, carbapenem resistance has become a threat and resistance genes are spreading among species through mobile genetic elements. The dissemination of carbapenemases among P.aeruginosa is a serious public health concern due to its limited options for the treatment of bacterial infections. In this study, it was aimed to investigate the molecular epidemiology of 47 carbapenem resistant P.aeruginosa (CRPA) isolates derived from various clinical samples from the Central Laboratory Bacteriology Unit of Kocaeli University Research and Training Hospital between October 2021 and March 2023. The rates of resistance to the antibiotics, some carbapenemase and virulence genes, conjugative resistance plasmids, integron gene cassette contents and the clonal similarity of the isolates were investigated and then epidemiologically evaluated. In the study, identification of the bacterial isolates and their susceptibility to some antibiotics (imipenem, meropenem, aztreonam, amikacin, netilmicin, tobramycin, piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, ciprofloxacin and levofloxacin) were determined by the VITEK® 2 Compact automated system. Metallo-beta-lactamase (MBL) production of the isolates was demonstrated by the imipenem/meropenem-EDTA (IMP/MEM-EDTA) combined disc method. Conjugation experiments were performed by the broth mating method. Alkali lysis method was used in plasmid DNA isolations. Co-transferred antibiotic resistances in transconjugants were detected by disc diffusion method. Carbapenemase genes (blaIMP , blaVIM , blaNDM , blaKPC and blaOXA-48 ), integron gene cassettes (class 1 and class 2) and virulence genes (lasR and rhlR) were screened by specific polymerase chain reactions (PCRs). Clonal relationships of the CRPA isolates were investigated by evaluating the DNA f ingerprintings obtained from the ERIC (enterobacterial repetitive intergenic consensus)-PCR assay. The highest resistance rate of the isolates were to levofloxacin, while the lowest resistance rates were observed against tobramycin, gentamicin and amikacin. MBL production was detected in 25 (53.2%) isolates. In conjugation experiments, 12 (25.5%) isolates were detected to harbour conjugative resistance plasmids. In 90% of the CRPA isolates, lasR and rhlR biofilm genes (encoding for the transcriptional activator protein) were detected by PCR. The blaVIM gene was detected in six (12.8%) isolates. The blaNDM gene was detected in five (10.6%) isolates and the blaOXA-48 gene was detected in three (6.4%) isolates. The blaKPC and blaIMP genes were not detected in CRPA isolates. It was determined that two (16.6%) of the isolates that carried the blaVIM gene, one (8.3%) carried the blaNDM gene and one (8.3%) carried the blaOXA-48 gene contained conjugative plasmids.In integron-specific PCRs, intI1 gene was positive in 39 (82.9%) isolates, while class 1 integron gene cassettes were detected in 24 isolates (51%). IntI1 positive six isolates were found to harbour class 1 integron gene cassettes-bearing conjugative plasmids. Class 2 integrons were not found in the CRPA isolates. Dendrogram analysis of ERIC-PCR patterns showed that there was no clonal similarity between the CRPA isolates and the isolates did not spread by cross-contamination. As a result, it has been observed that most of the CRPA isolates which have the potential to form biofilms, are highly resistant to other antibiotic groups other than carbapenems and can co-transfer some resistances (ceftazidime, cefepime, ciprofloxacin, levofloxacin, piperacillin-tazobactam) with conjugative resistance plasmids. It is thought that it would be useful to follow molecular epidemiology in the resistance gene reservoirs of these strains which have the potential to cause epidemics in the clinical arena.