Search Results (314)

Scientific evidence has suggested, in most cases, that nullity of the GSTM1 and GSTT1 genes is associated with worse pathological outcomes and viral infections. In this sense, the main objective of this work was to determine the genotypic frequencies of GSTM1 and GSTT1 polymorphisms in individuals with HIV and to establish a possible relationship with CD4+ T lymphocyte count. This was a cross-sectional study, with a quantitative approach, composed of 182 HIV-positive patients. To detect GSTM1 and GSTT1 polymorphisms by the multiplex polymerase chain reaction (PCR), oral mucosa samples were collected. Regarding genotypic frequencies, GST nullity was high in the population, being 97.5% and 97.6%, respectively, for GSTM1− and GSTT1−. Although there was no association between the GST polymorphism and the viral load and CD4+ T lymphocyte counts at diagnosis, when related to the current CD4+ count, the isolated and combined null alleles, GSTT1 (ORadj: 0.219; p = 0.004), GSTM1 (ORadj: 0.219; p = 0.004), and GSTM1/T1 (ORadj: 0.219; p = 0.004), were defined as factors favorable to a minimum CD4+ T lymphocyte count of 350 cells. Therefore, this study demonstrated a probable relationship between the GSTT1 and GSTM1 genetic polymorphisms and HIV. Full article

Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), Mycoplasma bovis (M.bovis), Pasteurella multocida (PM), Mannheimia haemolytica (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes. Full article

Cutaneous adverse reactions (CARs) are common complications of epidermal growth factor receptor (EGFR) inhibitor therapy, with papulopustular eruptions and paronychia being the most frequent. Growing scientific evidence implies that Staphylococcus aureus is involved in the pathogenesis of these reactions. This observational prospective study characterized 42 S. aureus strains isolated from CARs, analyzing antibiotic resistance, biofilm formation, soluble virulence factors, and virulence/resistance genes using multiplex polymerase chain reaction (PCR). S. aureus was identified in 90% of lesions; in 33% of cases, nasal and skin isolates were genetically identical. High resistance rates were noted for penicillins (85%) and tetracyclines (57%), while all strains remained susceptible to fluoroquinolones, vancomycin, and rifampicin. All isolates formed biofilms, and DNase/esculinase production significantly correlated with CAR severity. An enzymatic score based on these markers was associated with an 18-fold increased risk of severe reactions. Genotypically, clfA and clfB were prevalent (85.7%), while exotoxin genes were less common. These findings support a key role for S. aureus in exacerbating CARs via antibiotic resistance, biofilm production, and the expression of virulence factor. Additionally, we emphasize the role of routine microbial screening—including nasal swabs—and therapy guided by antibiograms. Furthermore, the enzymatic score may further be validated as a predictive biomarker. Full article

Background/Objectives: Meningitis due to the species Streptococcus is a severe central nervous system infection caused by various microorganisms belonging to Streptococcus spp. Its accurate identification is critical for effective clinical management. This study aimed to identify Streptococcus spp. causing meningitis in Greece over a nine-year period using PCR and sequencing-based methods. Methods: A total of 189 clinical samples, collected between 2015 and 2024 from patients suffering from meningitis and/or septicemia, were analyzed by the use of a combination of multiplex polymerase chain reaction (PCR) assays and tuf gene sequencing for further species identification. Results: Sample analysis identified 70 samples as S. pyogenes (18.52%) (GAS) and S. agalactiae (18.52%) (GBS), while 119 (62.96%) were recorded as non-typable Streptococcus spp. Further analysis using sequencing methods revealed that the most frequent Streptococcus spp. belonged to the mitis group (42.86%) and the pyogenic group (20.17%). A higher prevalence was observed in children aged 0–14 years old and adults over 50 years old. Conclusions: This study highlights the use of molecular diagnostics in identifying other Streptococcus spp., providing insights into age-related susceptibility and epidemiological trends. Future studies are needed to explore the pathogenic role of the identified Lactococcus spp. Full article

This study explored the distribution and genetic characteristics of respiratory pathogens in outpatients with acute respiratory infections (ARIs) in Yangon, Myanmar, during the 2023 rainy season. Among 267 patients who tested negative for influenza, RSV, and SARS-CoV-2 using rapid diagnostic tests, 84.6% were positive for at least one pathogen according to a multiplex polymerase chain reaction (PCR) assay, the BioFire^® FilmArray^® Respiratory Panel 2.1. The most common viruses detected were rhinovirus/enterovirus (RV/EV) at 37.8%, respiratory syncytial virus (RSV) at 22.4%, and human metapneumovirus (hMPV) at 10.0%. These pathogens co-circulated mainly from July to September, with RV/EV consistently predominant. Symptom comparison among RV/EV-, RSV-, and hMPV-infected patients showed similar clinical features, though fever was more common in hMPV cases. Among RV/EV-positive patients, 59.3% had single infections, while 40.7% experienced co-infections, especially with RSV and adenovirus. Genotyping identified 28 types from five species, primarily RV-A and RV-C, which were genetically diverse. One EV-D68 case was also found, emphasizing its potential risk. This study underscores the genetic diversity and clinical impact of RV/EV and stresses the importance of ongoing molecular surveillance in Myanmar’s post-COVID-19 context to inform effective public health responses. Full article

Duck enteritis virus (DEV), goose parvovirus (GPV), and muscovy duck parvovirus (MDPV) all have similar symptoms after infection, such as severe diarrhea, which seriously affects the healthy development of the waterfowl industry. Hence, it is important to devise a rapid and precise assay for the detection of these three viruses. In this study, a TaqMan probe-based multi-quantitative polymerase chain reaction (qPCR) assay was developed and optimized. Three specific primers and probes were designed according to the conserved regions of UL6 of DEV, REP of GPV, and VP1 of MDPV, respectively. DEV demonstrated a detection limit of 11.6 copies, GPV detected a limit of 95 copies, and MDPV showcased a detection limit of 14.8 copies. The correlation coefficient is greater than 0.99, and the amplification efficiency is 89% to 93%. These results indicate that the multiplex qPCR assay has high sensitivity, specificity, and stability. Of the 215 clinical samples used in this study, 33 tested DEV positive, 25 tested GPV positive, and 24 tested MDPV positive. Overall, the assay established in the current study presents a rapid, efficient, specific, and sensitive tool for of detecting DEV, GPV, and MDPV. Full article

Parainfluenza virus (PIV) infections contribute to the overall childhood morbidity from acute respiratory illness, yet virus-specific epidemiologic data are lacking across many regions globally. Here, we describe the clinical manifestations, seasonality, and meteorologic associations with PIV infections in Ecuadorian children. Between July 2018 and July 2023, we documented demographic and clinical information from children younger than 5 years seen in a single public health clinic with signs and symptoms consistent with an acute respiratory infection. Nasopharyngeal swabs collected at study enrollment underwent multiplex polymerase chain reaction-based diagnostic testing (Biofire FilmArray v. 1.7™). Regional meteorological data from the same period were provided by Ecuador’s Instituto Nacional de Meteorologia e Hidrologia. Parainfluenza viruses were detected in 9% of the 1251 enrolled subjects. PIVs were most frequently detected between March and July, with no change in seasonality following SARS-CoV-2 pandemic onset. Clinical manifestations of PIV infections included non-specific upper respiratory illness (82%), laryngotracheitis (3%), and bronchiolitis (11%). Events of PIV detection were negatively associated with ambient temperature and rainfall. Our findings highlight the contribution that PIVs play in the morbidity associated with pediatric medically attended outpatient respiratory tract infection and provide new insights into the seasonal epidemiology of PIV infections in coastal Ecuador. Full article

Introduction: The transforming growth factor beta (TGF-β) signaling pathway plays a critical role in cellular processes, including maintaining vascular integrity and regulating vascular remodeling. Aneurysm rupture is associated with pathological changes in the arterial wall. Aims: We aimed to investigate the gene expression of transforming growth factors (TGFB1, TGFB2, TGFB3) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of patients with unruptured intracranial aneurysms (UIAs) and ruptured intracranial aneurysms (RIAs), and from a control group. Additionally, we evaluated serum levels of TGF-β1, TGF-β2, and TGF-β3 and analyzed their associations with various risk factors, including sex, age, aneurysm size, number, shape, smoking, and hypertension. Materials and Methods: The study group consisted of patients diagnosed with intracranial aneurysms (IAs) who were eligible for embolization at the Department of Neurosurgery, Clinical Hospital of the Medical University of Bialystok. The control group consisted of healthy volunteers, recruited from the employees of the Clinical Hospital of the Medical University of Bialystok. Expression levels were assessed using quantitative real-time polymerase chain reaction techniques in PBMCs. Serum concentrations of TGF-β isoforms were evaluated using a multiplexed bead-based immunoassay. Results: Among 32 patients, 24 had unruptured intracranial aneurysms (UIAs), including 18 women and 6 men, while 8 presented with ruptured intracranial aneurysms (RIAs), evenly distributed between women and men (4 each). The mean age of the patients was 53 years (range: 24–71 years). The control group consisted of 20 healthy volunteers, 14 females and 6 males, with a mean age of 51 years (range: 24–71 years). The expression of TGFB1 was significantly higher in the IA versus C group, but TGFB3 expression was significantly higher in the RIA versus C group. The serum level of TGF-β1 and TGF-β3 was significantly higher in the RIA versus UIA group. Serum TGF-β1 levels were higher in men and individuals < 60 years of age. Positive correlations were observed between serum TGF-β1, TGF-β3 and aneurysm size, with significantly higher TGF-β3 levels in patients with giant aneurysms. Conclusions: Our study highlights the distinct roles of TGFB1 and TGFB3 in aneurysm pathophysiology, identifying TGFB1 as a molecular contributor to aneurysm formation and TGFB3 with rupture. Increased serum TGF-β1 and TGF-β3 concentrations could serve as promising noninvasive parameters for assessing the risk of aneurysm rupture. Further research with larger cohorts is needed to define cut-off values and validate the method, enabling the use of blood TGF-β levels as a tool for clinical decision-making. Full article

Background: If used in the right clinical context, PCRs carry great potential in rapidly diagnosing various infectious diseases. Objectives: We aimed to evaluate the clinical performance of four novel multiplex real-time PCR (qPCR) assays in the direct detection of pathogens in whole blood, cerebrospinal fluid, respiratory specimens, and stool samples. Methods: Spiked negative clinical specimens were used for the evaluation. Clinical samples for the comparative assessment of culture and molecular analyses were simultaneously examined. RINA^TM robotic nucleic acid isolation system and Bio-Speedy^® multiplex qPCR panels (Bioeksen R&D Technologies, Turkey), and the LightCycler^® 96 Instrument (Roche, USA) were used for the molecular testing. Results: No qPCR assays produced positive results for the samples spiked with the potential cross-reacting pathogens. The limit of detection (LOD) of the assays changed with the use of 10 and 100 pathogens/mL sample based on the target and sample type. The relative sensitivity and specificity of the assays were, respectively, 82% and 94% for blood, 97.1% and 99.3% for blood culture, 94% and 98% for stool, 96% and 97% for CSF, and 97% and 96% for respiratory specimens. Conclusions: The panels evaluated allow the direct molecular analysis of 10 samples from four clinical syndromes on the same run in 3 h with high clinical performance. The number and variety of samples in a single run enable healthcare providers to rapidly and efficiently diagnose and treat various infections. Full article

The proliferation of harmful cyanobacteria, particularly Microcystis, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic Microcystis in freshwater environments. We targeted the secA gene, specific to the Microcystis genus, and the mcyA gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected Microcystis at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring Microcystis dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms. Full article

Escherichia coli (E. coli) represents a significant etiological agent of colibacillosis in poultry, resulting in considerable economic losses for the global poultry sector. The present study aimed to determine molecular characterization, antibiotic resistance, and biofilm formation of E. coli strains isolated from diseased broilers from four provinces of China. A total of 200 tissue samples were collected from the intestine, liver, crop, heart, and spleen and processed for microbiological examination. Molecular detection of E. coli strains, virulence genes, and serotypes was performed using polymerase chain reaction (PCR). Antibiotic susceptibility testing and biofilm formation were assessed using disk diffusion and 96-well microtiter plate assays. The study retrieved 68% (136/200) of E. coli strains from collected samples. Most of the E. coli strains were resistant to enrofloxacin (56%), followed by cefepime (54%), amoxicillin/clavulanate (52%), streptomycin (50%), ampicillin (48%), clindamycin (47%), kanamycin (41%), polymyxin B (37%), tetracycline (35%), sulfamethoxazole/trimethoprim (33%), ceftazidime (31%), meropenem (4.7%), and florfenicol (2.9%). Similarly, the E. coli strains tested positive for at least one virulence gene and specific serotypes. Among these, O145 was the most prevalent serotype, identified in 22 isolates (16.2%), followed by O8 (12.5%), O102 (11.8%), and O9 (11.0%). The tsh gene (10.2%) was the most prevalent virulence gene. This study found that 47.1% of E. coli strains were biofilm-producing, with 62.5% exhibiting weak biofilm production, 29.7% mild biofilm production, and 7.8% strong biofilm production. Similarly, 24.2% of the E. coli strains were avian pathogenic E. coli strains due to the presence of five or more virulence genes, specifically tsh, ompA, fimC, iss, fyuA, and astA, in a single strain by multiplex PCR. The present study recommends continuous surveillance and effective control measures to reduce the burden of avian pathogenic E. coli-related infections in poultry. Full article

Multiplex Polymerase Chain Reaction (PCR) has significantly impacted the field of infectious disease diagnostics, offering rapid and precise identification of bacterial and fungal pathogens. Unlike traditional culture methods, which may take days to yield results, multiplex PCR provides diagnostic insights within hours, enabling faster, targeted antimicrobial therapy and reducing the delay in treating critical infections like sepsis. The technique’s high sensitivity and broad pathogen coverage make it ideal for both single and polymicrobial infections, improving outcomes across respiratory, bloodstream, and bacterial/fungal infections. However, multiplex PCR is not without challenges; initial high costs and the need for specialized training can limit its adoption, especially in low-resource settings. This review discusses the clinical advantages and limitations of multiplex PCR, highlighting its influence on diagnostic accuracy, antimicrobial stewardship, and the global fight against antimicrobial resistance (AMR). Furthermore, recent innovations in multiplex PCR, such as digital PCR and portable devices, are explored as potential tools for expanding access to rapid diagnostics worldwide. Full article

Background/Objectives: Mycoplasma pneumoniae (M. pneumoniae), traditionally associated with mild community-acquired pneumonia in school-aged children, has experienced a delayed resurgence following the COVID-19 pandemic. The epidemiological and clinical characteristics of M. pneumoniae pneumonia in children within the context of this global resurgence have not been well established in Romania. Materials and Methods: This retrospective, single-center study analyzed children diagnosed with M. pneumoniae pneumonia who were hospitalized in the pulmonology department of “Grigore Alexandrescu” Emergency Hospital for Children in Bucharest from March to December 2024. Clinical, laboratory, and radiographic data were extracted from hospital records. M. pneumoniae infection was confirmed through polymerase chain reaction (PCR) multiplex panel detection or specific IgM antibody levels ≥ 10 AU/mL. Results: The final analysis included 63 patients who met the inclusion criteria. The cohort’s median age [IQR] was 12.6 [8–15] years, with 11.1% (n = 7) under 6 years old. The radiographic findings revealed a predominance of right lung involvement (52.4%, n = 33, p = 0.03) and a significantly higher prevalence of alveolar infiltrates compared to interstitial patterns (88.9%, n = 56, p < 0.001). Antibiotic choice did not significantly affect hospitalization duration. Pleural effusion emerged as a common complication, occurring in 27% (n = 17) of patients and associated with elevated admission leukocyte counts (p = 0.007). Rare extrapulmonary manifestations included meningoencephalitis (1.6%, n = 1) and reactive infectious mucocutaneous eruption (3.2%, n = 2). Notably, co-infections with other respiratory pathogens did not extend hospital stays. Conclusions: This study contributes to the evolving global epidemiological profile of M. pneumoniae infections in the post-pandemic era. It establishes a foundation for future multi-center analyses aimed at monitoring the changing epidemiology and clinical presentations of M. pneumoniae infections in pediatric populations. Full article

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R² values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. Full article

The distribution of oral pathogens is influenced by genetic background, diet, socioeconomic status, and racial factors. This study aimed to assess the distribution and characteristics of oral pathogens based on blood glucose levels in a South Korean population. This cross-sectional, retrospective study included subjects from 17 health promotion centers in 13 South Korean cities between November 2021 and December 2022. Real-time multiplex PCR was used to detect 10 periodontitis-related pathogens, 6 dental caries-related pathogens, and 1 dental caries-protective bacterium. The most prevalent periodontitis-related pathogens were Parvimonas micra (97.6%), Porphyromonas endodontalis (96.8%), and Treponema socranskii (95.0%). Among dental caries-related pathogens, Streptococcus sanguinis and Veillonella parvula were found in all subjects. The prevalence of periodontitis-related pathogens was higher in males, while pathogens related to periodontitis and dental caries were more prevalent in older individuals. In the diabetes group, Aggregatibacter actinomycetemcomitans, red and orange complexes, and Streptococcus mutans were more prevalent. The relative amount of S. sanguinis was lower, while V. parvula was higher in individuals with diabetes mellitus. The prevalence and composition of oral pathogens vary by sex, age, and blood glucose levels. Diabetic individuals showed a pathogenic community structure linked to increased risks of periodontitis and dental caries. Full article