Polymerase Chain Reaction Method Research Articles

Authentication of the Apis cerana honey with the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method

Honey adulteration has been on the rise with the increasing demand for high-quality honey, and the authentication of A. cerana honey is a major problem that the current bee industry needs to solve. The aim of this research was to establish the proofreading enzyme-mediated probe cleavage combined with ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method for the authentication of Apis cerana honey. The LMTIA primers were designed to target the specific 18S rRNA sequence of A. cerana, and the conditions for Proofman-LMTIA amplification were optimized. The specificity and sensitivity of the method were determined and compared with that of the droplet digital polymerase chain reaction (PCR). The optimal temperature of the established Proofman-LMTIA method was 62 °C. The method can specially to detect the A. cerana gene without cross-reactivity with the genomes from other animal and plant species, and the amplification reaction can be finished within 20 min with the sensitivity of 10 pg A. cerana genomic DNA, which was more sensitive than the droplet digital PCR method. The obtained results demonstrate that the established Proofman-LMTIA procedure was specific, sensitive, and rapid, and can be used for authentication of A. cerana honey.

Association of leukocyte telomere length with cardiac conduction disease and sick sinus syndrome

Abstract Background Cardiac conduction disease (CCD) and sick sinus syndrome (SSS) may be related to a degenerative process. Leukocyte telomere length (LTL) is associated with the aging process and age-related degenerative diseases. We aimed to investigate the association between LTL and the risks of CCD/SS, and to explore whether LTL is related with cardiovascular imaging and electrocardiographic (ECG) changes. Methods This is a population-based cross-sectional study of UK Biobank participants without history of CCD/SSS recruited from 2006 to 2010. LTL was measured using a quantitative polymerase chain reaction method. Cardiovascular measurements were derived from cardiovascular magnetic resonance using machine learning. The median (IQR) duration of follow-up was 12.0 (11.3-12.7) years. The associations of LTL with imaging and ECG measurements and incident CCD/SSS were evaluated by multivariable regression models. Results Among the 412,452 white participants, 188,891 (45.8%) were male, with a mean age of 57.3 years. Longer LTL was independently associated with positive cardiac remodeling, including higher left ventricular mass, larger atrial volume, and increased atrial stroke volumes. However, no significant associations were observed with ECG findings including PR interval and P-wave duration. 11991 (2.6%) participants developed CCD and 984 (0.2%) developed SSS. Longer LTL was not associated with younger age. However, longer LTL was not associated with a lower risk of incident CCD (LTL fourth quartile vs. first quartile: hazard ratio, 0.98; 95% CI, 0.97-1.0; P = 0.057) or SSS (LTL fourth quartile vs. first quartile: hazard ratio, 0.98; 95% CI, 0.92-1.05; P = 0.554). Conclusions In this cross-sectional study, longer LTL was associated with a larger atrial size with better atrial function in middle age, but not with ECG changes. However, longer LTL was not associated with a lower risk of incident CCD and SSS.

Comparative study of two laboratory techniques for the detection of HLA-B27 in patients with axial spondyloarthritis: a cross-sectional analysis

BackgroundThe diagnostic and prognostic relevance of Human Leukocyte Antigen B-27 (HLA-B27) in Axial Spondyloarthritis (AxSpA) is undeniable, with 70% of Ankylosing Spondylitis (AS) patients carrying the B27 gene, contrasted with a mere 4.35% in the general population. Flow cytometry (FC) and Polymerase Chain Reaction (PCR) have emerged as the predominant techniques for routine HLA-B27 typing. While various studies have compared these methods, none have catered to the unique characteristics of the Brazilian demographic. Therefore, this research aims to compare FC and PCR in a Brazilian cohort diagnosed with AxSpA.MethodsAn analytical cross-sectional study was undertaken involving 62 AxSpA outpatients from a Brazilian University Hospital. Both FC and PCR-SSP assays were utilized to ascertain HLA-B27 typing. The outcomes (either confirming or refuting the allele’s presence) underwent rigorous scrutiny. Agreement between the methodologies was assessed using the kappa statistic. A p-value of < 0.05 was deemed statistically significant.ResultsOf the participants, 90.3% (n = 56) were HLA-B27 positive according to FC, while 79% (n = 49) were identified as positive using the PCR method. FC exhibited a sensitivity rate of 98% paired with a specificity of 38.5%. The Positive Predictive Value for FC stood at 85.7%, and the Negative Predictive Value was 83.5%. Consequently, the overall accuracy of the FC method was gauged at 85.5%. A kappa coefficient of κ = 0.454 was derived.ConclusionsFC demonstrated noteworthy sensitivity and satisfactory accuracy in HLA-B27 detection, albeit with a reduced specificity when contrasted with PCR-SSP. Nevertheless, given its cost-effectiveness and streamlined operation relative to PCR, FC remains a pragmatic option for preliminary screening in clinical practice, especially in low-income regions. To optimize resource allocation, we advocate for a refined algorithm that initiates by assessing the relevance of HLA-B27 typing based on Choosing Wisely recommendations. It then leans on FC, and, if results are negative yet clinical suspicion persists, advances to PCR. This approach aims to balance diagnostic accuracy and financial prudence, particularly in regions contending with escalating medical costs.

Development of a TaqMan polymerase chain reaction detection method for the precise identification and quantification of an attenuated Eimeria maxima vaccine strain in poultry

Avian coccidiosis, a parasitic disease prevalent in poultry, is caused by Eimeria species and leads to significant economic losses. The use of attenuated live oocyst vaccines has been adopted as an alternative to the use of anticoccidial drugs. However, the accurate detection and differentiation of vaccine strains from virulent ones remain challenging. Therefore, this study presents a novel TaqMan polymerase chain reaction (PCR) detection method that offers enhanced sensitivity, specificity, and reproducibility compared with traditional PCR techniques. Through whole-genome resequencing and bioinformatics analysis, we identified a molecular marker gene, Em_marker6, with a unique 21-base pair deletion specific to the Eimeria maxima attenuated vaccine strain. Optimized primers and probes targeting this marker enabled rapid quantification cycle value achievement and high fluorescence intensity. The standard curve’s slope of −3.540 and correlation coefficient of 0.9971 confirmed precise quantification capabilities. The TaqMan PCR method detected as few as 30 plasmid DNA copies and 50 oocysts per reaction, outperforming traditional PCR techniques by an order of magnitude. No cross-reactivity was observed with other E. maxima wide-type strains or common intestinal pathogens, ensuring the exclusive detection of the E. maxima EMPY vaccine strain. Weekly testing over 3 weeks demonstrated minimal variability, indicating robust consistency in the method’s application. Testing on 61 clinical samples revealed a 57.38% positivity rate for E. maxima species and 13.11% for the vaccine strain. The Em_marker6 gene exhibited genetic stability across multiple generations, confirming the detection method’s robust stability for the attenuated E. maxima vaccine strain. This study significantly advances the field of avian coccidiosis research and control by providing a valuable tool for monitoring vaccine purity and preventing inadvertent infections in vaccinated flocks, aligning with global efforts to curb antibiotic use in animal feed.

Ventilation improvement for effective protection of healthcare workers in negative pressure airborne infectious isolation room from viral aerosols

A negative pressure airborne infectious isolation room (AIIR) is the primary healthcare air contamination control system used for the treatment of severe respiratory infectious patients. Effects of the ventilation system configuration and conditions on airflow pattern, aerosol distribution and ventilation performance were investigated using computational fluid dynamics (CFD). The field measurement by SARS-CoV-2 environmental surface test was also conducted. The cycle threshold values from transcription polymerase chain reaction (RT-PCR) method showed inverse relation to the simulated number of particles trapped on the surfaces indicating a good agreement. Modification of the present AIIR to have alignment between air inlet and outlet where the aspect ratio of the air outlet, Width (W): Height (H) = 1:1 (Improved case: IC#1) showed a 78 % reduction of aerosol concentration in healthcare workers (HCWs) zones. Aerosol concentrations were increased when the openings of the air outlet were enlarged. Addition of air outlet led to large swirling air, resulting in more aerosols being trapped and suspended in the air. Results suggested that AIIR with alignment air inlet on the ceiling and air outlet at the wall over the patient's head with W:H = 1:1 be the most suitable configuration to maximize the ventilation performance and minimize exposure risk to aerosolized viral infection for HCWs. Air change rate plays a more important role than the differential pressure on the removal efficiency. The differential pressure value should be at least −2.5 Pa and the air supply rate 12 ACH for effective protection of HCWs in the negative pressure AIIR.

3'-Daidzein Sulfonate Sodium Protects against Glutamate-induced Neuronal Injuries by Regulating NMDA Receptors.

It was previously found that 3'-Daidzein Sulfonate Sodium (DSS) exhibits protective effects on Cerebral Ischemia-Reperfusion Injury (CI/RI). This study aimed to explore the underlying molecular mechanisms involved in the neuroprotective effects of DSS against ischemic stroke. In this study, rats with transient middle cerebral artery occlusion (tMCAO) were used as an in vivo model, whereas PC12 cells treated with glutamate alone and rat primary cortical neurons treated with the combination of glutamate and glycine were used as in vitro models. Cell viability and lactate dehydrogenase (LDH) release were used to evaluate cell injury. Cell apoptosis was determined by flow cytometry. Quantitative polymerase chain reaction (qPCR), Western blotting, and immunofluorescent staining methods were used to determine the mRNA expressions and protein levels and location. It was found that DSS significantly suppressed the impaired viability of PC12 cells induced by glutamate. DSS also increased cell viability while reducing the LDH release and apoptosis in primary cortical neurons injured by glutamate and glycine. In addition, DSS decreased GluN2B subunit expression while enhancing the expressions of GluN2A subunit and PSD95 in tMCAO rats' brains. This study demonstrated that DSS protects against excitotoxic damage in neurons by regulating the expression of NMDA receptors and PSD95 in the brain with cerebral ischemia-reperfusion injury. Our findings provide experimental evidence for the potential clinical administration of DSS in ischemic stroke.

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Identification and genotyping of Chikungunya virus using reverse transcription polymerase chain reaction and restriction fragment length polymorphism methods

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RT-PCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

Review of Detection Limits for Various Techniques for Bacterial Detection in Food Samples.

Foodborne illnesses can be infectious and dangerous, and most of them are caused by bacteria. Some common food-related bacteria species exist widely in nature and pose a serious threat to both humans and animals; they can cause poisoning, diseases, disabilities and even death. Rapid, reliable and cost-effective methods for bacterial detection are of paramount importance in food safety and environmental monitoring. Polymerase chain reaction (PCR), lateral flow immunochromatographic assay (LFIA) and electrochemical methods have been widely used in food safety and environmental monitoring. In this paper, the recent developments (2013-2023) covering PCR, LFIA and electrochemical methods for various bacterial species (Salmonella, Listeria, Campylobacter, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli)), considering different food sample types, analytical performances and the reported limit of detection (LOD), are discussed. It was found that the bacteria species and food sample type contributed significantly to the analytical performance and LOD. Detection via LFIA has a higher average LOD (24 CFU/mL) than detection via electrochemical methods (12 CFU/mL) and PCR (6 CFU/mL). Salmonella and E. coli in the Pseudomonadota domain usually have low LODs. LODs are usually lower for detection in fish and eggs. Gold and iron nanoparticles were the most studied in the reported articles for LFIA, and average LODs were 26 CFU/mL and 12 CFU/mL, respectively. The electrochemical method revealed that the average LOD was highest for cyclic voltammetry (CV) at 18 CFU/mL, followed by electrochemical impedance spectroscopy (EIS) at 12 CFU/mL and differential pulse voltammetry (DPV) at 8 CFU/mL. LOD usually decreases when the sample number increases until it remains unchanged. Exponential relations (R2 > 0.95) between LODs of Listeria in milk via LFIA and via the electrochemical method with sample numbers have been obtained. Finally, the review discusses challenges and future perspectives (including the role of nanomaterials/advanced materials) to improve analytical performance for bacterial detection.

Isolated ocular sarcoidosis

A rare clinical case of a patient with isolated sarcoidosis of the eyes is presented. Sarcoidosis is a polyethological disease, the morphological feature of which is the formation of epithelioid cell granulomas in various organs and tissues, which is accompanied by a violation of their structure and function. Both congenital and adaptive immune systems are involved in the pathogenesis of sarcoidosis, with damage to lymph nodes, lungs, skin, organ of vision, etc. Risk factors for the development of sarcoidosis have not been definitively established. Environmental factors and genetic predisposition play a crucial role in the development of the disease. The above clinical observation demonstrates the polymorphism of the clinical picture of isolated ocular sarcoidosis. In addition to standard ophthalmic examination methods, the genetic method of reverse transcription and quantitative polymerase chain reaction (RT-PCR, English qRT-PCR) was also used to determine the expression profile of signature microRNAs as potential diagnostic markers of sarcoidosis of the organ of vision.An increase in the expression level of hsa-miR-455-3p, hsa-miR-4663, hsamiR68063p, hsa-miR-450b-5p, hsa-miR-6716-3p, hsa-miR-5000-5p, hsa-miR150-5p can be considered as potential diagnostic biomarkers of sarcoid lesion of eye structures. With sarcoidosis, the involvement of the lacrimal gland is possible. In the presented clinical observation, this was indicated by the results of a functional examination, sonographic signs: revealed multiple linear tubular hyperechoic structures and a decrease in blood flow velocity in the ocular and lacrimal arteries, as well as an increase in the expression activity of hsa-miR150-5p (p &lt; 0.05). In order to prevent possible damage to the ocular surface due to the development of lacrimal gland dysfunction, it is advisable to prescribe local tear replacement therapy with hyaluronic acid preparations. Such effective drugs include tear substitutes of the Optinol® line: “Optinol® Express Moisture” and “Optinol® Deep Moisture”.

Subgingival microbiome in periodontal disease and comorbid pathology (meta-analysis)

The problem of chronic periodontitis (CP) is actively discussed due to the recognition of the fact that periodontal microbial damage is closely related to a number of systemic diseases and probably plays an important role in the occurrence of comorbid pathology. The aim of the meta-analysis was to characterize the composition of the subgingival microbiome and to determine the peculiarities of the formation of associations of the new periodontal pathogen Filifactor alocis with other I and II order periodontal pathogenic bacteria, as well as with the commensal bacteria colonizing this biotope. The study presents data of patient examination with obligatory use of polymerase chain reaction methods and sequencing of 16S rRNA genes in 1529 healthy individuals and 2394 patients with CP, 136 patients with CP and concomitant atherosclerosis, 258 patients with CP and concomitant type 2 diabetes mellitus. It was confirmed that the basis of the oral microbiome under normal circumstances is composed of representatives of microaerophilic streptococci, corynebacteria, lactobacilli, as well as representatives of the Veillonella and Sphingobacterium genera. 16S sequencing and bioinformatic analysis allowed us to specify the taxonomic place of the new pathogen F. alocis, as well as representatives of normal microbiota in CP and comorbid somatic pathology.

A novel nanoplasmonic-based diagnosis platform: Advances and emerging technologies

The development of a rapid, simple, inexpensive, and sensitive nucleic acid assay is critical in ultrafast diagnostic systems to prevent the rapid spread of novel infectious agents. However, current representative diagnostic methods, such as the polymerase chain reaction (PCR), are bulky, expensive, complex, and time-consuming. The plasmonic photothermal effect of plasmonic materials is widely used in biological applications because of excellent properties, such as strong optical properties and enhanced local electromagnetic field. PCR methods using plasmonic nanostructures are used to overcome the limitations of conventional PCR. Therefore, plasmonic photothermal-based PCR exhibit a rapid ramping rate, ultrafast amplification, high sensitivity, cost-effectiveness, and compactness, which can be used as a point-of-care device in situ. This study reviewed the mechanisms of plasmonic PCR and various materials, such as organic, inorganic, and hybrid materials, used in PCR. Furthermore, the properties, light source, amplification efficiency, and monitoring method of classified plasmonic materials were compared. This review introduced the disease diagnosis application using plasmonic PCR and evaluated the prospect of potential future applications as next-generation PCR.

Performance evaluation of loop-mediated isothermal amplification, polymerase chain reaction and real-time polymerase chain reaction methods to detect Neisseria gonorrhoeae among symptomatic patients from India.

Background: Neisseria gonorrhoeae is one of the most important causative organisms in causing sexually transmitted infections. The clinical presentation of gonorrhoea mimics the symptoms of other sexually transmitted infections, and a proper diagnosis of the same is therefore crucial in patient management. The current study intended to compare different in-house molecular methods: that is, conventional PCR, real-time PCR, and LAMP assay for detection of N. gonorrhoeae. Methods: A total of 163 samples were collected from 145 patients who presented with urethral and vaginal discharge. Collected samples were processed for culture on GC agar base, and three different molecular diagnostic tests (conventional PCR, real-time PCR, and LAMP assay) were performed simultaneously on all the samples. Results: Culture of N. gonorrhoeae was positive in 17 out of 21 (80.9%) swab samples. With culture as the gold standard method, conventional and real-time PCR had a sensitivity of 94.1%, whereas the sensitivity of the LAMP assay was found to be 88.2%. All three methods had a specificity of 100%. In addition to swab samples, evaluation of urine samples by different molecular methods yielded a good concordance with a kappa value of 0.85 by conventional PCR and real-time PCR showing a perfect level of agreement, while the LAMP assay was found to have a substantial level of agreement. Conclusion: LAMP assay had a comparable diagnostic accuracy to other molecular methods for the detection of N. gonorrhoeae and can be used as a point-of-care test in resource-limited settings.

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

High frequency of multidrug-resistant Escherichia coli from cattle in the Cerrado and Pantanal biomes of Brazil

The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100%) exhibited some phenotypic resistance to antimicrobials and 67.62% exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28%), followed by toxigenic Escherichia coli (ETEC) (11%), enterohemorrhagic Escherichia coli (EHEC) (8%) and enteropathogenic Escherichia coli (EPEC) (2%). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33%), ETEC + EHEC (15%) and ETEC + EPEC (3%). The STEC pathotype (30%) was also found more frequently in the Pantanal, followed by EHEC (12%), ETEC (9%) and EPEC (6%). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.

Evaluation of Malaria Rapid Diagnostic Test Performance and pfhrp2 Deletion in Tanzania School Surveys, 2017.

As part of malaria nationwide monitoring and evaluation initiatives, there is an increasing trend of incorporating malaria rapid diagnostic tests (mRDTs) in surveys conducted within primary schools to detect malaria parasites. However, mRDTs based on the detection of histidine-rich protein 2 (HRP2) are known to yield false-positive results due to persistent antigenemia, and false-negative results may result from low parasitemia or Plasmodium falciparum hrp2/3 gene deletion. We evaluated diagnostic performance of an HRP2 and pan-parasite lactate dehydrogenase (HRP2/pLDH) mRDT against polymerase chain reaction (PCR) for detection of P. falciparum among 17,051 primary school-age children from eight regions of Tanzania in 2017. According to PCR, the prevalence of P. falciparum was 19.2% (95% CI: 18.6-19.8). Using PCR as reference, the sensitivity and specificity of mRDT was 76.2% (95% CI: 74.7-77.7) and 93.9% (95% CI: 93.5-94.3), respectively. Test agreement was lowest in low transmission areas, where true-positive mRDTs were outnumbered by false-negatives due to low parasitemia. Discordant samples (mRDT-negative but PCR-positive) were screened for pfhrp2/3 deletion by real-time PCR. Among those with a parasite density sufficient for analysis, pfhrp2 deletion was confirmed in 60 samples, whereas pfhrp3 deletion was confirmed in two samples; one sample had both pfhrp2 and pfhrp3 deletions. The majority of samples with gene deletions were detected in the high-transmission Kagera region. Compared with mRDTs, PCR and other molecular methods offer increased sensitivity and are not affected by pfhrp2/3 deletions, making them a useful supplement to mRDTs in schools and other epidemiological surveys.

Molecular detection of Plasmodium DNA in goats in Kulon Progo, Indonesia

The Plasmodium parasites transmitted through the bites of infected Anopheles mosquitoes cause malaria in humans. Despite extensive research on the malaria parasite in ungulates, its occurrence in goats in Indonesia, particularly in the Kaligesing and Girimulyo border areas, has yet to be reported. This study aimed to investigate the presence of malaria parasites in goats from these regions. We collected 81 blood samples from goats of different ages and sexes. Blood samples were drawn from the jugular vein and preserved using EDTA tubes. We evaluated the overall clinical status of the goats by monitoring their rectal temperature and capillary refill time (CRT). Microscopic analysis was carried out by observing blood smears under a microscope, but the Plasmodium parasite was not detected. Next, we employed a molecular method to detect Plasmodium DNA using a nested polymerase chain reaction (PCR) method that targeted the cytochrome C oxidase subunit I (COI) gene. The results confirmed the presence of Plasmodium DNA in 5 out of the 81 samples tested. The obtained data were analyzed descriptively, and Wilson’s confidence interval method was used to calculate confidence limits for the sample proportions. This study provides the first molecular detection of previously undocumented malaria parasites in goats within the Girimulyo region of Kulon Progo, Yogyakarta, Indonesia. Identifying Plasmodium sp. using molecular techniques highlights the need for continued surveillance and management strategies to prevent the spread of malaria in animals and humans.

Detection of trichomonads in patients with lung cancer and transcription analysis on the response of human pulmonary epithelial cells to Trichomonas tenax invasion

IntroductionLung cancer is one of the most prevalent malignancies worldwide. Substantial research has illuminated the intricate interplay between microorganisms and human health, revealing their role in disease regulation. Trichomonads is a flagellated protozoan in the human cavity and have been previously identified as a pathogen associated with pneumonia, contributing to tissue chronic inflammation and carcinogenesis. MethodsNested polymerase chain reaction methods were employed to scrutinize the prevalence of trichomonads in the bronchovesicular fluid of patients diagnosed with lung cancer. Subsequently, the influence of Trichomonas tenax invasion on lung cancer cells was elucidated through proliferation assays, migration assays, and transcription analysis. ResultsBronchoalveolar fluid samples from lung cancer patients yielded positive nested PCR results for eight out of twenty-seven samples. Seven of these samples were identified as Trichomonas tenax, while one was identified as Tetratrichomonas spp. Our findings revealed a significant upregulation of pathways associated with carcinogenesis, including cellular proliferation, migration, and drug resistance, in response to T. tenax invasion. ConclusionsThis study underscores the importance of recognizing the presence of trichomonads and the influence of T. tenax invasion on host responses to respiratory diseases. The identified pathways implicated in cancer development may pave the way for developing targeted treatment strategies for pulmonary diseases. These findings hold promise for informing and improving the precision of therapeutic interventions in the context of pulmonary ailments.

Verification of quantitative polymerase chain reaction method for monitoring minimal residual disease in chronic myeloid leukemia

Verification of quantitative polymerase chain reaction method for monitoring minimal residual disease in chronic myeloid leukemia