Polymerase Chain Reaction Results Research Articles

Assessment of fungal species in some spices using molecular study with detection of their mycotoxins using chromatography analysis.

Spices are food flavouring agents that are highly used in Iraq. However, they may be contaminated by toxicogenic fungi and subsequent production of mycotoxins. The aim of this study was to investigate the contamination of commonly used spices with fungi using polymerase chain reaction (PCR) assay and to detect fungal mycotoxin using high-performance liquid chromatography. Thirty-five spices (seven samples from each black pepper, red pepper, turmeric, cumin and ginger) were cultured on an appropriate medium to identify various fungi species. Later on, the toxigenicity of Aspergillus flavus and Aspergillus niger was determined using a PCR assay. The fungal mycotoxins, including aflatoxins and ochratoxins, were then determined through a high-performance liquid chromatography using the validated Quick, Easy, Cheap, Effective, Rugged, and Safe (QuECHERS) method. Aspergillus species were the predominantly isolated fungi, followed by Penicillium and Fusarium. The PCR results indicate the high toxigenicity of A. flavus as 85.7% of the strains had aflQ/aflR genes and 79% had PKS15KS/PKS15C-MeT genes. Regarding mycotoxin contamination in spices, the highest rates of aflatoxins and ochratoxins were found in black pepper (5.913 μg/kg) and red chilli (6.9055 μg/kg), respectively. Spices are susceptible substrates for the growth of mycotoxigenic fungi. Thus, regular effective surveillance and quality control procedures are highly recommended.

Mycobacterium avium subsp. paratuberculosis in Wild Boar (Sus scrofa) in Portugal.

Paratuberculosis, or Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The agent was also found in wild mammals such as wild boar (Sus scrofa); however, the role of wild mammals in the epidemiology of MAP is unclear. During the research period, 941 free-ranging wild boar (S. scrofa) legally hunted in two locations in the central-eastern region of Portugal were examined. Ninety-seven wild boars exhibited one or more gross lesions and were tested for the presence of Mycobacterium avium subsp. paratuberculosis using acid-fast staining, mycobacterial culture, polymerase chain reaction (PCR), and histopathological examination. Forty-five animals (46.4%, 95% CI: 36.5-56.3%) were identified as infected, as indicated by positive results in culture and/or PCR. The findings revealed that the most significant risk factor was being a juvenile compared to yearlings and adults (OR = 10.2, 95% CI: 2.2-48.0). Based on our results, 37.9% (n = 11) of the infected animals were considered suitable for human consumption. Our findings offer novel insights into mycobacterial infections in wild boar populations in Portugal and suggest that wild boar could be a source of human infection if zoonotic potential is considered.

Detection of Carbapenem Resistance Using the Genotypic and Phenotypic Methods in Klebsiella pneumoniae

Aim: This study aimed to detect the carbapenem resistance of the Klebsiella pneumoniae strains, isolated from clinical specimens with genotypic and phenotypic methods. Material and Methods: A total of 87 Klebsiella pneumoniae strains whose carbapenem resistance was determined by disc diffusion method were included in the study. Carbapenemase was investigated using the combined disk method and polymerase chain reaction (PCR). Results: The evaluation of the PCR results demonstrated that OXA was detected in 60 (68.9%) samples, NDM was detected in 20 (22.9%), OXA + NDM in 5 (5.7%), and KPC was detected in 1 (1.1%) out of 87 clinical samples. Carbapenemase was not detected in one specimen with the PCR method. The results were found compatible with the combined disc test results for all isolates which were detected as only OXA, NDM, and KPC type carbapenemase positive. In 5 (5.7%) strains in which the co-existence of NDM and OXA type carbapenemases was detected by PCR, the combined disc method detected only OXA type carbapenemase. Conclusion: The combined disk method is inadequate in the presence of strains that have multiple carbapenemases, and also have OXA which is the most frequently detected carbapenemase in our hospital. EUCAST recommends verification by other methods in the presence of OXA-48. Genotypic methods can be used for confirmation testing. The detections of strains with NDM, multiple carbapenemases, and the first detection of KPC were striking in the study. Monitoring the spread of these strains in the hospital will be necessary for infection control.

Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.

Molecular diversity of Klebsiella pneumoniae clinical isolates: antimicrobial resistance, virulence, and biofilm formation

One of the mechanisms responsible for antibiotic resistance in Klebsiella pneumoniae is the enzymes produced by the bacteria; another important mechanism is the ability to form biofilm. In this study, antibiotic resistance, genes associated with virulence, and biofilm-forming properties of K. pneumoniae strains were investigated. A total of 100 K. pneumoniae isolates were obtained from different clinical samples identified by Matrix-Assisted Laser Desorption/Ionization time-of-flight Mass Spectrometry. Antimicrobial susceptibility testing was performed with the Phoenix 100 apparatus. The biofilm forming properties of strains were determined by the microtiter plate method. For molecular analysis, genes encoding the carbapenemase enzyme (bla OXA-48, bla NDM-1, bla IMP, and bla VIM) and biofilm-related genes (treC, luxS, mrkA, and wza) were investigated by polymerase chain reaction (PCR). While 76% of clinical isolates were resistant to three or more antimicrobials, 24% were classified as non-multidrug resistant (non-MDR). When biofilm-forming capacities of clinical isolates were tested, it was determined that the resistant-isolates produced 59.2% strong biofilm, and susceptible-isolates produced 12.5% strong biofilm. According to PCR results, carbapenemase genes were determined as follows: bla OXA−48-70%, bla NDM-49%, and bla KPC-19%, bla OXA-48/bla NDM/bla KPC-12%, bla OXA-48/bla NDM-26%, and bla OXA-48/bla KPC-4%. The biofilm-associated genes in bacterial isolates were determined as follows: luxS-98%, treC-94%, mrkA-88%, and wza-15%. In addition, Hierarchical Clustering Tree and Heatmap analysis revealed an association between isolates that lacks resistance genes and isolates lacks biofilm-formation related genes that were included in MDR or non-MDR classes. As a result, biofilm should be considered in the treatment of MDR infections, and therapy should be planned accordingly. In addition, pursuing the data and genes of antibiotic resistance is significant for combating resistance.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81kcal/mol, while the docking energy for meropenem was - 4.92kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28kcal/mol, compared with - 4.60kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16µg/mL, whereas levofloxacin-resistant strains had MIC values of 8µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2h under the action of 8µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Comparison of the real-time polymerase chain reaction and xpert Mycobacterium tuberculosis/rifampicin assay for the detection of Mycobacterium tuberculosis

Abstract Background A serious issue with world health is tuberculosis (TB). Annual estimates from the World Health Organization (WHO) put the number of incident cases at 9.6 million. The aim of this work was to compare between real-time polymerase chain reaction (PCR) and GeneXpert in recognition of Mycobacterium tuberculosis (MTB) with special reference to quantification of MTB and to speed of detection of the organism. Materials and Methods Thirty individuals participated in this prospective clinical quasi-interventional trial with clinical data and chest radiography suggestive of pulmonary tuberculosis. Participants were split into three groups equally: group I (control group) was zielneilsen stain (ZN) stain positive and culture positive, group II were ZN stain negative and culture positive and group III were ZN stain negative and culture negative. Each participant was exposed to a plain chest radiography and a complete blood picture. Results Real-time PCR result was significantly correlated with loss of appetite and weight in group III. There were negative correlations between PCR and mediastinal lymphadenopathy in group I. In group I GeneXpert was significantly correlated with night sweat and fever, breathlessness and cavitary lesion. In group III GeneXpert was significant correlated with site of lesion and night sweat and fever. Conclusions For TB patients, the PCR test is crucial in order to provide prompt diagnosis and therapy. When comparing AdvanSure tuberculous /non tuberculous mycobacterium (TB/NTM) real-time PCR with Xpert MTB/rifampicin (RIF) assay, the latter is more advantageous due to its test-method simplicity and quicker than real-time PCR ability to simultaneously identify rifampin resistance. However, its sensitivity for specimens with an acid-fast bacilli (AFB) smear negative but a positive culture is less than AdvanSure TB/NTM real-time PCR. With special reference to the quantification of MTB, TB/NTM real-time PCR is a quantitative test but Xpert MTB/RIF is semiquanitative.

Implications of Measles Inclusion by Commercial Syndromic Polymerase Chain Reaction Panels - United States, May 2022-April 2023.

Syndromic polymerase chain reaction (PCR) panels are used to test for pathogens that can cause rash illnesses, including measles. Rash illnesses have infectious and noninfectious causes, and approximately 5% of persons experience a rash 7-10 days after receipt of a measles, mumps, and rubella (MMR) vaccine. MMR vaccine includes live attenuated measles virus, which is detectable by PCR tests. No evidence exists of person-to-person transmission of measles vaccine virus, and illness does not typically result among immunocompetent persons. During September 2022-January 2023, the Tennessee Department of Health received two reports of measles detected by syndromic PCR panels. Both reports involved children (aged 1 and 6 years) without known risk factors for measles, who were evaluated for rash that occurred 11-13 days after routine MMR vaccination. After public health responses in Tennessee determined that both PCR panels had detected measles vaccine virus, six state health departments collaborated to assess the frequency and characteristics of persons receiving a positive measles PCR panel test result in the United States. Information was retrospectively collected from a commercial laboratory testing for measles in syndromic multiplex PCR panels. During May 2022-April 2023, among 1,548 syndromic PCR panels, 17 (1.1%) returned positive test results for measles virus. Among 14 persons who received a positive test result and for whom vaccination and case investigation information were available, all had received MMR vaccine a median of 12 days before specimen collection, and none had known risk factors for acquiring measles. All positive PCR results were attributed to detection of measles vaccine virus. Increased awareness among health care providers about potential measles detection by PCR after vaccination is needed. Any detection of measles virus by syndromic PCR testing should be immediately reported to public health agencies, which can use measles vaccination history and assessment of risk factors to determine the appropriate public health response. If a person recently received MMR vaccine and has no risk factors for acquiring measles, additional public health response is likely unnecessary.

PCR-based study on viral pathogens circulation among cervids in the Moscow region

A molecular survey of selected viruses in free-ranging cervids was conducted in 15 different districts of Moscow region. Samples were collected from 178 game animals including 144 moose (Alces alces), 19 roe deer (Capreolus capreolus) and 15 deer without species information. Nasal swabs and tissue samples including parts of the nasal septum, upper tracheal rings, lung, heart, liver, kidneys and pooled organ samples were tested using polymerase chain reaction (PCR). Samples were studied for pestiviruses, herpesviruses, coronaviruses, group A rotaviruses, adenoviruses, hepatitis e and parainfluenza type 3 virus. None of the samples were positive for Bovine Coronavirus and SARS-COV-2, hepatitis E virus and parainfluenza type 3 virus. PCR results were positive for bovine herpesviruses (5.05%), pestiviruses (0.56%), rotaviruses (1.68%). DNA of a new adenovirus, presumably causing a mild course of animal respiratory disease, was detected in samples of 6 animals (3.37%). In conclusion, the conducted studies have shown that game animals of the Moscow region can be a natural reservoir of cattle viruses, and this must be taken into account when planning and organizing measures for the control and eradication of such notifiable diseases as bovine viral diarrhoea and infectious bovine rhinotracheitis. Monitoring studies and general disease surveillance of wild animal populations provide additional information on the epidemiology of infectious diseases in the region and allow timely measures to be taken to protect wild animals, domestic animals and the public.

A cost-effective and field-deployable sensing system for chip-integrated detection of bacteria with the naked eye

To tailor the broadly applied polymerase chain reaction (PCR) technology to point-of-care settings, an intuitive, time-saving, and cost-effective method to determine the DNA amplification results has significant practical potential. Herein we present an economical and field-deployable sensing system that enables naked-eye readout of PCR results. The proposed system is powered by an amplification enhancer to greatly enhance the positive-to-background ratio and a palm-sized readout device to assist on-site applications. High specificity, as well as single-copy sensitivity, was achieved in detecting Salmonella enterica and other bacterial pathogens. Using the proposed system, we identified the pathogenic bacterial strains in artificially contaminated food samples easily, quickly, and reliably. Also crucial for point-of-care testing, this naked-eye readout system applies to microfluidic platforms with the sample volumes scaled down to 1 μL. The time savings, portability, and cost-effectiveness of the established sensing system make it transformative for on-chip bacteria detection in point-of-care and field settings, especially in resource-limited areas.

Bacterial Flora and Treatment Strategies in Women With Escherichia coli Urinary Tract Infections.

Introduction This study explores the intricate relationship between bacterial flora and the occurrence of Escherichia coli (E. coli) infections in gynecological patients. It aims to provide insights into the various treatment strategies used to effectively manage bacterial pathogens, especially E. coli infections. By conducting a comprehensive analysis of the bacterial flora in gynecological patients, the study highlights the notable presence of E. coli, promptingfurther investigation into the factors that contribute to its colonization. The objective of the study is to comprehensively investigate and detect urinary tract infections (UTIs) specifically caused by E. coli among gynecological patients. The study aims to delve into bacterial flora prevalence, antibiotic resistance patterns, and potential virulence factors. Through this analysis, the study intends to identify effective strategies for rapid detection and diagnosis of UTIs caused by E. coliby utilizing advanced microbiological and molecular techniques. Furthermore, the study aims to formulate and propose a strategic treatment approach with a particular emphasis on selecting appropriate antibiotics to reduce the risk of severe infections and associated complications. Materials and methods The methodology employed in this study included the isolation and characterization of bacterial strains from clinical samples obtained from gynecological patients. A total of 52 urine specimens were collected from patients with complaints of infection in the urinary tract and infertility. These samples underwent both preliminary and confirmatory microbiological analysis, such as gram staining, biochemical confirmation test, and antibiotic susceptibility, and further proceeded with the multiplex polymerase chain reaction (PCR) technique. The results of PCR and antibiotic susceptibility revealed the specific gene involvement and resistant characteristics of E. coli. Results The findings revealed a total of 32 specimens positive for E. coli, of which 10 patients had infertility complaints and 22 patients had UTIs. The preliminary test, gram staining, showed the gram-negative bacilli E. coli, and the nutrient agar plate revealed smooth circular translucent colonies; MacConkey agar showed pink-colored lactose-fermented colonies; and the blood and chocolate agar plates showed grayish white moist gamma-hemolytic colonies. The biochemical confirmation of E. coli resulted in positive for indole and methyl red tests and negative for Voges-Proskauer and citrate utilization tests. The multiplex PCR analysis confirmed the E. coli strains with the presence of two target genes,stx2d and stx2e. Conclusion To summarize, this study offers valuable insights into the bacterial flora of gynecological patients impacted by E. coli infections, which provides a foundation for the development of precise and efficient treatment strategies. The results emphasize the importance of personalized treatment approaches that consider both the microbiological characteristics of the infection and the evolving landscape of antibiotic resistance. The implication of this research extends to enhancing clinical outcomes and alleviating the burden of E. coli infections in gynecological settings.

Investigation of gut microbiota diversity according to infectious agent in pediatric infectious acute gastroenteritis in a Korean university hospital

BackgroundAcute gastroenteritis (AGE) is a common cause of pediatric morbidity and mortality worldwide. AGE can cause an imbalance in the intestinal microbiota. This study aimed to investigate the diversity of the gut microbiome in Korean children hospitalized for infectious AGE at a university hospital. MethodsA total of 23 stool samples from patients aged 5 months to 11 years with AGE were analyzed. Thirteen convalescent stool samples were collected 1 month after discharge. Multiplex polymerase chain reaction (PCR) for the five viruses and 16 bacteria-specific AGE pathogens (PowerChek Multiplex Real time PCR Kit, Seoul, Korea), and 16s rRNA sequencing (Illumina MiSeq Sequencing system, Illumina, USA) were performed. ResultsAccording to the results of multiplex PCR for causative pathogens, the microbiome taxonomic profile (MTP) of the gut microbiome in three groups of AGE, norovirus AGE (n = 11), Campylobacter AGE (n = 7) and Salmonella AGE (n = 5) was compared. The phylum Actinobacteria was significantly more abundant in the norovirus AGE (P = 0.011), whereas the phylum Proteobacteria was significantly more abundant in Salmonella AGE (P = 0.012). Alpha diversity, which indicates species richness and diversity, showed no statistical differences. However, beta diversity, representing the similarity in MTP between norovirus, Campylobacter, and Salmonella AGE, was significantly different (P = 0.007). In convalescence, compared with their corresponding AGE samples, the phylum Firmicutes; and the lower taxa Christensenellaceae (P = 0.0152) and Lachnospiraceae (P = 0.0327) were significantly increased. ConclusionsIn pediatric AGE, the type of infectious agent can affect the diversity and dominance of gut microbiota in pediatric patients. Furthermore, healthy gut bacteria increased during the period of 1 month after infection, allowing a return to a healthy state without causing long-term dysbiosis.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction(PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having anHPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Escherichia coli and their potential transmission of carbapenem and colistin-resistant genes in camels

BackgroundCamels harbouring multidrug-resistant Gram-negative bacteria are capable of transmitting various microorganisms to humans. This study aimed to determine the distribution of anti-microbial resistance among Escherichia coli (E. coli) isolated from the feces of apparently healthy camels in Egyptian abattoirs. Additionally, we sought to characterize Shiga toxin-producing E. coli (STEC) strains, assess their virulence potential, and investigate the possibility of camels spreading carbapenem- and colistin-resistant E. coli.Methods121 fecal swaps were collected from camels in different abattoirs in Egypt. Isolation and identification of E. coli were performed using conventional culture techniques and biochemical identification. All isolates obtained from the examined samples underwent genotyping through polymerase chain reaction (PCR) of the Shiga toxin-encoding genes (Stx1 and Stx2), the carbapenemase-encoding genes (blaKPC, blaOXA−48, blaNDM, and blaVIM), and the mcr genes for mcr-1 to mcr-5.ResultBacteriological examination revealed 75 E. coli isolates. PCR results revealed that one strain (1.3%) tested positive for Stx1, and five (6.6%) were positive for Stx2. Among the total 75 strains of E. coli, the overall prevalence of carbapenemase-producing E. coli was 27, with 7 carrying blaOXA48, 14 carrying blaNDM, and 6 carrying blaVIM. Notably, no strains were positive for blaKPC but a high prevalence rate of mcr genes were detected. mcr-1, mcr-2, mcr-3, and mcr-4 genes were detected among 3, 2, 21, and 3 strains, respectively.ConclusionThe results indicate that camels in Egypt may be a primary source of anti-microbial resistance (AMR) E. coli, which could potentially be transmitted directly to humans or through the food chain.

Comparison of clinical outcomes of patients with serial negative surveillance cultures according to a subsequent polymerase chain reaction test for carbapenemase-producing Enterobacterales

Carbapenemase-producing Enterobacterales (CPE) are of serious concern worldwide due to high morbidity and mortality. To evaluate the impact of the result of a subsequent polymerase chain reaction (PCR) test for carbapenemase after serial negative surveillance cultures on positive culture conversion in patients with three consecutive negative surveillance cultures for CPE, and to identify risk factors for conversion. A retrospective study of patients with positive CPE cultures on CHROMagar KPC medium was performed in a Korean tertiary hospital from October 2018 to December 2022. PCR for blaKPC, blaNDM, blaIMP, blaVIM, blaGES, and blaOXA-48 was performed after three consecutive negative rectal swab cultures. Clinical characteristics and outcomes of patients were compared according to whether follow-up PCR was positive (CNPP) or negative (CNPN). Of 1075 patients with positive CPE cultures, 150 (14.0%) yielded three consecutive negative rectal swab cultures. Of these, 50 (33.3%) were CNPP, and 100 (66.7%) were CNPN. Risk factors associated with a positive PCR result on multivariate analysis were: age, central venous catheter, and Escherichia coli infection. CNPP patients were more likely to have positive culture conversion for CPE than CNPN patients (39/44 (88.6%) vs 21/50 (42.0%), P<0.001). In multivariate analysis, independent risk factors for culture conversion were: a positive PCR result after surveillance cultures, diabetes mellitus, central venous catheter, and Klebsiella pneumoniae. CNPP patients have higher rates of culture conversion than CNPN patients, and a follow-up PCR test after serial negative surveillance cultures is useful in deciding whether or not to discontinue contact precautions.

First report of co-infections of Marek's disease virus and chicken infectious anaemia virus in poultry flocks in Nigeria

Marek's disease (MD) and chicken infectious anaemia (CIA) are viral immunosuppressive diseases of poultry caused by the MD virus (MDV) and CIA virus (CIAV) respectively. Despite vaccination against MD, the incidence of the disease in vaccinated poultry flocks in Nigeria persists. However, underlying factors like co-infection with CIAV have not been investigated in the country. This study was designed to investigate possible co-infections of MDV and CIAV in poultry flocks in Nigeria. In 2016, tumorous tissue samples were collected from suspected cases of MD at necropsy in Jos, Plateau State, Nigeria. The samples collected were fixed in formalin for histopathological examination, genomic DNA was extracted from a second part and analysed by polymerase chain reaction (PCR), targeting the meq and VP1 genes of the MDV and CIAV, respectively. The histology results revealed that the cutaneous and proventricular lymphomas were characterized by large numbers of mononuclear cellular infiltrates admixed with heterophils. The PCR results revealed that MDV was detected in 66.7% (16/24), CIAV in 45.8% (11/24), and co-infections of MDV and CIAV were detected in 45.8% (11/24) of the samples analysed. In addition, co-infections of MD and CIA were recorded in 100% (6/6) and 27.7% (5/18) of broilers and layer/pullet’ samples respectively. Phylogenetic analysis of the meq gene sequences revealed that the Nigerian MDV clusters with very virulent MDV from Egypt and Italy. While, CIAV sequences were genotype II and genotype III and clustered with CIAVs from Cameroon and China. This is the first report of co-infections of MD and CIA in Nigeria.

Insulin Ameliorates Neurodegeneration in an Experimental Animal Model of Brain Aging by a Decrease of GFAP Expression and Apoptosis Inhibition

Background: Aging is one of the factors that will cause gradual changes in brain tissue and its destruction. One of the protective factors against aging is insulin. Objectives: Our study investigated insulin's neuroprotective effect by preventing oxidative stress-mediated neuronal damage. Methods: A total of 48 adult male NMRI (the Naval Medical Research Institute) mice were divided into 2 groups: control and insulin. Insulin was administered intraperitoneally (IP) for 8 weeks to the control group and for 12 weeks to the insulin group. In this study, animals were treated with insulin, and insulin's role in the aging process was evaluated. Then, brain tissue was extracted for evaluation of oxidative stress by assessment of glutathione disulfide (GSH) and reactive oxygen species (ROS) levels, the expression of the glial fibrillary acidic protein (GFAP) as a biomarker for astrogliosis, stereological study, and the real-time polymerase chain reaction (PCR) technique evaluated the level of apoptosis biomarker. Results: The results of stereological parameters show that the volumes of the brain cortex and the number of neurons improved in the insulin group by aging as compared to the control group. The results of real-time PCR showed that the expression level of apoptotic markers decreased in the insulin group compared to the control group. In the insulin group, GSH levels were higher than in the control group, and the control group produced more ROS than the insulin group. As compared with insulin, the control group expressed more GFAP protein and increased the number of glial cells by aging. Conclusions: The results showed that insulin could have neuroprotective effects against aging changes and reduce apoptosis and cell death.

Cat Scratch Disease of the Breast/Axilla: Recognition of a Rare Disease and Approaches for Differential Diagnosis.

Cat scratch disease rarely presents as a breast or axillary mass mimicking carcinoma both clinically and radiologically. Diagnosing breast/axillary cat scratch disease is challenging due to its rarity and nonspecific findings. Here, we reported 2 patients with breast cat scratch disease and reviewed 14 patients with cat scratch disease involving breast/axilla from the past 30 years. It mainly affects women (median age: 48), consistently presenting as axillary lymphadenopathy, and demonstrates ipsilateral breast mass in half of patients (50%, 8/16). The breast mass was most commonly located in the upper outer quadrant (88%, 7/8), indicating the possibility of disease extension from axillary adenopathy. Around half of patients (56%, 9/16) reported cat exposure. Histologically, most patients (93%, 14/15) presented as necrotizing granulomas, with characteristic stellate-shaped necrosis in 5 patients. Although pathologic differential diagnoses between cat scratch disease and cancer are straightforward, distinguishing cat scratch disease from other granulomatous mastitis poses diagnostic challenges. Silver stains should be included in the diagnostic workup panel when highly suspecting cat scratch disease clinically. However, they were only able to highlight the causative microorganism in 54% (7/18) patients, and the gram stain was negative in all 12 tested patients. In contrast, polymerase chain reaction (PCR) for the causative microorganism was consistently positive in all 3 tested patients, while serologic test confirmed diagnosis in 85% (11/13) patients; 1 patient with negative serology showed a positive PCR result. Therefore, upfront PCR tests with or without serologic study should be considered to confirm the diagnosis of cat scratch disease in a timely manner.

Molecular prevalence and associated risk factors of Cryptosporidium spp. infection in dairy cattle in Khon Kaen, Thailand

Background and Aim: Cryptosporidium spp. are important parasites in the small intestines of humans and animals, particularly cattle. The aim of this study was to estimate the molecular prevalence and associated risk factors of Cryptosporidium infection in dairy cattle in five districts of Khon Kaen province, Thailand, and to identify Cryptosporidium spp. Materials and Methods: From July 2020 to October 2021, 296 fecal samples were collected from three groups of dairy cattle: Calves aged &lt;3 months, calves aged 3 months–1 year, and calves aged &gt;1 year. Cryptosporidium spp. were detected by polymerase chain reaction (PCR) amplifying the 18s RNA gene. Both genus-specific and species-specific primers were used to identify Cryptosporidium confirmed by DNA sequencing. Age, house floor type, and water trough type were evaluated as risk factors. We analyzed all associated risk factor information using the logistic regression test in the Statistical Package for the Social Sciences. Results: PCR results showed that 40 (13.51%) out of 296 samples were positive for Cryptosporidium spp., including Cryptosporidium bovis (57.50%) and Cryptosporidium ryanae (2.50%). There was a significant association between Cryptosporidium incidence, cattle age, and house floor type (p = 0.05). National Center for Biotechnology Information Basic Local Alignment Search Tool displayed 99.48%–100% nucleotide similarity of each Cryptosporidium spp. isolate with references recorded on GenBank. Conclusion: C. bovis and C. ryanae are commonly found in dairy cattle, especially calves, in Khon Kaen, Thailand, and the incidence was associated with age and house floor type. A molecular technique may be influential for species identification. The results of the present study would provide useful information for veterinarians and animal owners to understand better Cryptosporidium spp. and how to manage farms properly. Keywords: bovine cryptosporidiosis, Cryptosporidium, dairy cattle, molecular prevalence.