Advancing IBDV diagnostics: a one-step multiplex real-time qRT-PCR for discriminating between vvIBDV and non-vvIBDV viruses, including the newly emerged IBDV variant.

The very virulent infectious bursal disease virus (vvIBDV) induces an acute, highly contagious and immunosuppressive disease in younger chicken causing massive economic losses globally. A major challenge in the field's clinical diagnosis is distinguishing gross lesions caused by vvIBDV from those induced by classic IBDV (cIBDV), commonly used as live attenuated vaccines. This study introduces a one-step multiplex real-time PCR assay designed to distinguish between vvIBDV and non-vvIBDV viruses. Via simultaneously targeting the VP2 sequence for vvIBDV detection and the VP1 sequence for non-vvIBDV identification, including classic, American variant and the recently emerged novel variant IBDV (nvarIBDV), the assay's specificity was validated against common avian viral diseases and nonspecific IBDV strains without any observed cross-reactions. It effectively differentiated between vvIBDV and non-vvIBDV field samples, including nvarIBDV, as confirmed by genotyping based on VP2 sequencing. The assay demonstrated a limit of detection ranging from 1.9×1010 to 103 DNA copies for vvIBDV-VP2, 9.2×1010 to 103 DNA copies for classic strains, and 1.2×1011 to 104 DNA copies for nvarIBDV in VP1 detection of non-vvIBDV. In conclusion, this study presents a specific, sensitive, and straight forward multiplex real-time PCR assay.

Employment of Spore-Forming Probiotics to Combat Persister Cells of Staphylococcus Epidermidis.

Background: In this study, spore-forming probiotics were employed to eradicate Staphylococcus epidermidis biofilms and the presence and expression of genes involved in stress response was examined. Methods: Polymerase chain reaction (PCR) assay was used to detect rpoS, relA and mazF genes in S. epidermidis ATCC 12228. Biofilm production was investigated by microtiter plate (MTP) assay. 100X minimum inhibitory concentration (MIC) of gentamycin was used to induce persister cells in planktonic and biofilm bacterial cells. The expression of rpoS, relA, and mazF genes was assessed at different time intervals of 2, 8, and 24 h using real-time PCR assay. Then, dilutions of 1, 0.5, and 0.25 µg/ml of the supernatant of Bacillus coagulans culture was used to eradicate the persister cells and the number of colonies was determined. Results: Persister cells of S. epidermidis were formed after 7 h in planktonic and 5 h in the biofilm structure after exposure to 50 µg/ml of gentamycin. The expression of mazF and rpoS in biofilm structure and the expression of rpoS and relA in persister cells were significantly higher compared to the control (p< 0.05). The number of persister cells showed a reduction of log 2.4 and log 0.8 after exposure to 1 and 0.5 µg/ml B. coagulans supernatant, respectively, but no reduction was observed at the concentration of 0.25 µg/ml. Conclusion: The results showed that the supernatant of probiotics containing their secretive metabolites can be used as a novel approach to combat persister cells.

Concurrent genotyping and expression of NLRP3 inflammasome in pityriasis versicolor patient's skin lesions.

The goal of this study is to investigate the impact of the rs35829419 SNP on the serum level of NLRP3, and to assess the relationship between NLRP3 and its SNP and vulnerability to Pityriasis versicolor. Pityriasis versicolor (PV) is one of the most frequent skin conditions linked to skin pigmentation changes. Malassezia plays a key role in the pathogenesis of PV. A case-control study, 50 patients with pityriasis versicolor and 44 healthy controls. Real-time PCR was used to genotype NLRP3 (rs35829419) and ELISA assay of NLRP3 levels in tissue samples. There was a significantly higher median NLPR3 levels in PV patients than controls. A significant predominance of A allele of Q 705 K was in patients than controls. The risk of having the disease in the presence of A allele is nearly 10 times than having C allele. In PV patients, there was a significant relationship between NLPR3 levels and Q 705 K genotypes with higher NLPR3 levels in AA genotype. A potential correlation between PV and the Q705K polymorphism, pointing to evidence of NLRP3 alteration in PV patients. The NLRP3 inflammasome may be an appropriate therapeutic target for Malassezia-associated skin disorders.

Clinical evaluation of a real-time PCR assay for diagnosis of Helicobacter pylori infection and antibiotic resistance.

OBJECTIVES: Helicobacter pylori (H. pylori) is a globally prevalent bacterium that increases the risk of developing various gastrointestinal diseases, including gastric adenocarcinoma. This study aimed to evaluate the performances of real-time PCR assay in detecting H. pylori infection, as well as clarithromycin and levofloxacin resistance, in both stool and gastric biopsy specimens. METHODS: Stool and gastric biopsy specimens were collected from patients within one to three days post-hospitalization. All patients were analyzed for H. pylori infection and resistance to clarithromycin and levofloxacin using a real-time PCR based molecular assay. RESULTS: 169 patients (83 males) with a mean age of 43.6±13.1 years were included in the study. The prevalence of H. pylori was 89.9% (152/169) in stool and 90.5% (153/169) in gastric biopsy samples. The molecular diagnostics employed in this study exhibited a sensitivity of 99.3% and a specificity of 100%, resulting in a diagnostic accuracy rate of 99.6%. Resistance to clarithromycin was 36.1% (61/169) in stool and 44.4% (75/169) in gastric biopsy samples. The molecular tests for clarithromycin resistance demonstrated a sensitivity of 96.8% and a specificity of 86.8%, with an overall diagnostic accuracy of 90.5%. Furthermore, resistance to levofloxacin was 22.5% (38/169) and 26.6% (45/169) in stool and gastric biopsy samples, respectively. The molecular test demonstrated a sensitivity of 80.9% and a specificity of 94.3%, resulting in a diagnostic accuracy of 90.5%. CONCLUSION: The implementation of real-time PCR-based screening for H. pylori infection and resistance to clarithromycin and levofloxacin in the stool may enhance the success rate of eradication therapy.

Multiplexed real-time PCR for the detection and differentiation of Klebsiella pneumoniae O-antigen serotypes.

Klebsiella pneumoniae has emerged as a global health threat due to its role in the spread of antimicrobial resistance and because it is a frequent cause of hospital-acquired infections and neonatal sepsis. Capsular and lipopolysaccharide (LPS) O-antigen polysaccharide surface antigens are major immunogens that are useful for strain classification and are candidates for vaccine development. We have developed real-time PCR reagents for molecular serotyping, subtyping, and quantitation of the most prevalent LPS O-antigen types (i.e., O1, O2, O3, and O5) of Klebsiella pneumoniae. We describe two applications for this O-typing assay: for screening culture isolates and for direct typing of Klebsiella pneumoniae present in stool samples. We find 100% concordance between the results of the O-typing assay and whole-genome sequencing of 81 culture isolates, and >90% agreement in O-typing performed directly on specimens of human stool, with disagreement arising primarily from a lack of sensitivity of the culture-based comparator method. Additionally, we find evidence for mixed O-type populations at varying levels of abundance in direct tests of stool from a hospitalized patient population. Taken together, these results demonstrate that this novel O-typing assay can be a useful tool for K. pneumoniae epidemiologic and vaccine studies.IMPORTANCEKlebsiella pneumoniae is an important opportunistic pathogen. The gastrointestinal (GI) tract is the primary reservoir of K. pneumoniae in humans, and GI carriage is believed to be a prerequisite for invasive infection. Knowledge about the dynamics and duration of GI carriage has been hampered by the lack of tools suitable for detection and strain discrimination. Real-time PCR is particularly suited to the higher-throughput workflows used in population-based studies, which are needed to improve our understanding of carriage dynamics and the factors influencing K. pneumoniae colonization.

Multifocal tuberculosis revealed by a sternal swelling in an immunocompetent child.

Tuberculosis (TB) is one of the most common pathogens of bacterial lung infections, especially in underdeveloped nations like Morocco, where the incidence of TB was 97 cases per 100 000 persons in 2019. Thanks to its national TB prevention and control plan, Morocco was able to achieve remarkable progress in the management of TB with an 80% reduction in the total number of patients diagnosed with TB between 1980 and 2018. The national plan also allowed us to reach and maintain a therapeutic rate above 86% since 2002. Sternal TB is a rare clinical condition accounting for 1% of all musculoskeletal TB cases. Due to its rarity and the lack of awareness of clinical presentations, the diagnosis of sternal TB can be quite complex. We describe the case of a 14-year-old Moroccan patient consulting in the Military Hospital Mohammed V-Rabat with central chest pain for 4 months which was not associated with breathing, physical exercise or eating. The patient also had a history of asthenia, fever and weight loss. A computed tomography scan of the chest showed a destructive lesion of the sternum. Afterward, a chirurgical biopsy was performed and enabled to confirm the microbiological diagnosis of TB with the realization of the real-time PCR. The antitubercular therapy was given to the patient who had complete resolution of symptoms. This condition should be included in the differential diagnosis of chronic chest pain that mimics costochondritis particularly in patients from endemic areas.

The employment of real-time polymerase chain reaction for analysis of canine meat in meatball products for halal authentication analysis.

Objective: Meatballs are a popular meat-based food consumed widely in Indonesian society. However, the issue of unethical substitution of halal meatballs with non-halal meats, particularly pork and canine meat (CM), has emerged. The existence of non-halal meats, including CM, in food products is prohibited in Islam, necessitating the development of reliable analytical techniques for their identification. In this study, we designed species-specific primers (SSPs) targeting the D-loop region of mitochondrial DNA for CM meatball product identification. Materials and Methods: The study was commenced by creating specific primers for canine DNA using Integrated DNA Technologies software and subsequently performing DNA isolation. The designed primers were then subjected to comprehensive evaluation using RT-PCR, including specification, linearity, limit of detection, efficiency, and repeatability. Results: The results indicated that the primer D-Loop 443 (forward: 5'-GGG ACA TCT CGA TGG ACTA ATG-3', reverse: 5'-GCG GTC ATA GAT GAG TGA TAG C-3') designed and validated in silico using primer-basic local alignment search tool nucleotide (BLAST) program from NCBI accurately identified canine DNA when the optimal annealing temperature was set at 57.5oC. The real-time PCR technique utilizing the D-loop 443 primer exhibited the ability to amplify canine DNA down to a minimum quantity of 100 pg, with an efficiency value of 91.8%, a correlation coefficient (R) of 0.990, and a precision value (RSD) of 0.30%. Conclusion: The SSP-based RT-PCR method developed is a versatile and efficient tool for detecting CM in meatballs. Its implementation helps maintain consumer trust and addresses concerns regarding the substitution of halal meats with non-halal alternatives.

Effective RNA isolation method for gram-positive and acid-fast bacteria: metamorphosed from conventional RNA isolation techniques.

The RNA-based study provides an excellent indication of an organism's gene expression profile. Obtaining high-yield and high-purity RNA from Gram-positive and acid-fast bacteria is difficult without high-end kits and facilities. We optimised effective and simple protocol for RNA isolation that is a combination of enzymatic, physical and chemical treatment to disrupt cells. We successfully isolated high quality intact total RNA with yields ranging from 23.13 ± 0.40 to 61.51 ± 0.27 µg and the 260/280 purity ratio of 1.95 ± 0.01 to 2.05 ± 0.01 from Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Mycobacterium smegmatis. These results represents a significantly enhanced yield and purity compared to other combination of techniques which we performed. Compared to previous studies the yield obtained by this method is high for the studied organisms. Furthermore the yielded RNA was successfully used for downstream applications such as quantitative real time PCR. The described method can be easily optimised and used for various bacteria.

Development of highly adaptable RT-PCR methods for identifying Delta and BA.1 variants in inactivated COVID-19 vaccines.

Background The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses a significant threat to human health and public safety. While next-generation sequencing (NGS) is capable of detecting and tracking new COVID-19 variants for disease diagnosis and prevention, its high cost and time-consuming nature limit its widespread use. In this study, our aim was to develop a highly adaptable and accurate RT-PCR method for identifying the Delta or BA.1 variants in inactivated COVID-19 vaccine. We devised three two-plex RT-PCR methods targeting specific mutation sites: S: Δ156-157, S: N211-, L212I, and S: Δ142-144, Y145D. The RT-PCR method targeting the S: Δ156-157 mutation site was able to distinguish the Delta variant from other COVID-19 virus strains, while the RT-PCR methods targeting the S: N211-, L212I or S: Δ142-144, Y145D mutation sites were able to distinguish the BA.1 variant from other COVID-19 virus strains. We separately validated these three two-plex RT-PCR methods, and the results demonstrated good linearity, repeatability, reproducibility, and specificity for each method. Moreover, all three methods can be applied in the production of SARS-CoV-2 variant inactivated vaccines, enabling the identification of Delta or BA.1 variants in virus cultures as well as in inactivated vaccine stocks. This study presents a systematic approach to identify COVID-19 variants using multiple RT-PCR methods. We successfully developed three two-plex RT-PCR methods that can identify Delta and BA.1 variants based on specific mutation sites, and we completed the validation of these three methods.

Real-time PCR assay to detect the novel Clade Ib monkeypox virus, September 2023 to May 2024.

Monkeypox virus (MPXV) is an emerging zoonotic pathogen with complex epidemiology necessitating rapid diagnosis and distinguishing between clades and subclades. The emerging Clade Ib lacks the genomic region used in the Clade I-specific assay from the Centers for Disease Control and Prevention. We report an MPXV real-time PCR to specifically detect Clade Ib. The assay demonstrated proficient sensitivity and specificity in 92 samples and can be included along other TaqMan-based assays to detect MPXV and distinguish between clades and subclades.

Adopting the 2023 CDC Early Testing for Perinatal Hepatitis C: Call to Action for Pediatric Primary Care Providers.

In the US, the burden of hepatitis C virus (HCV) infection is disproportionately high among young adults including pregnant persons, resulting in increased infections among children as perinatal transmission remains the main route of HCV infection in children. Hence, in 2020, the Centers for Disease Control and Prevention (CDC) recommended universal HCV screening during each pregnancy. HCV infection in infancy is usually asymptomatic, so the diagnosis entirely relies on testing of perinatally-exposed infants which, historically, included anti-HCV antibody testing at ≥ 18 months of age. However, nation-wide perinatal HCV testing rates have been suboptimal with significant loss to follow up. To address this problem, in 2023, the CDC introduced early single HCV RNA testing at 2-6 months of age with an alternative for HCV RNA testing up to 17 months of age if not previously tested. The high sensitivity and specificity of the HCV real-time PCR laid the grounds for this policy shift. In this review we highlight how these new CDC recommendations will enhance testing of infants and children and ultimately contribute to overall HCV elimination efforts. We also emphasize the role of all pediatric providers and obstetricians in implementing these new guidelines. Additionally, we offer our perspective and practical advice for testing of perinatally exposed infants and children. Currently, curative oral antivirals for HCV-infection treatment are approved for children ≥ 3 years of age. As pediatricians, advocating for children's wellness, it is our utmost duty to ensure that every child exposed to perinatal hepatitis C has been tested, diagnosed, linked to care, treated, and achieved cure.

Evaluation of a commercial Real Time PCR for clinical samples without RNA extraction for detection of SARS-CoV-2.

RT-PCR is the gold standard for diagnosis of COVID-19. All RT-PCR kits are based on RNA extraction from the clinical sample. There was a sudden increase in demand of these kits, both RNA extraction and COVID-19 RT-PCR kits during the pandemic. This sudden spurt in global demand created a situation of shortage of consumables, especially the RNA extraction kits. Hence, this study was carried out to evaluate and compare COVID-19 RT-PCR without RNA extraction step using buffer R3. Sensitivity, specificity and accuracy of RT-PCR kit without RNA extraction were 89.16 %, 100% and 89.6% respectively. This approach saved more than 50 % time compared to the RT-PCR kit with RNA extraction approach allowing enhanced daily sample processing capability. RT-PCR kit without RNA extraction help in managing a greater number of samples, reduces cost and turnaround time.

Development and Validation of a Novel Multiplex Real-Time PCR Assay for Rapid Detection of Carbapenemase Genes in Carbapenem-Resistant Enterobacterales Isolates and Clinical Samples.

Purpose: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods. Methods: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay. Results: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2×102 copies/mL to 8×102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing. Conclusion: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Quantitative method for measuring the proportion of bacterial cells expressing phase 1 or 2 of flagellin of Salmonella enterica serovar Typhimurium.

Salmonella enterica subsp. enterica is a major pathogen that causes zoonotic foodborne diseases worldwide. Some Salmonella serovars possess two antigenic phases for flagellin: phase 1 and 2. In Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellin is antigenically divided into "Hi" as phase 1 and "H1 or H2" as phase 2. Flagellin phase variation is regulated by inversion of hin gene. We focused on the inversion of hin and developed a real-time PCR system to quantitatively measure the proportion of bacterial cells expressing each phase of flagellin. In this study, we demonstrated that our newly developed real-time PCR system shows high quantitative accuracy and aligns with flagellin expression status. Furthermore, the newly developed real-time PCR system was applicable to various S. Typhimurium laboratory and field strains. This newly developed real-time PCR system has the potential to become a powerful tool for analyzing flagellin phase variation.

Validation of real-time PCR assays for detecting Plasmodium and Babesia DNA species in blood samples.

Malaria and babesiosis are global health threats affecting humans, wildlife, and domestic animals, particularly in Africa, the Americas, and Europe. Malaria can lead to severe outcomes, while babesiosis usually resembles a mild illness but can be severe and fatal in individuals with weakened immune systems. Swift, accurate detection of these parasites is crucial for treatment and control. We evaluated a real-time PCR assay for diagnosing five Plasmodium and three Babesia species from blood samples, assessing its sensitivity, specificity, and analytical performance by analyzing 46 malaria-positive and 32 Babesia spp-positive samples diagnosed through microscopy. The limit of detection for Plasmodium species ranged from 30 to 0.0003 copies/µL. For mixed infections, it was 0.3 copies/µL for P. falciparum/P. vivax and 3 copies/µL for P. malariae/P. knowlesi. Babesia species had a detection limit of 0.2 copies/µL. No cross-reactivity was observed among 64 DNA samples from various microorganisms. The assay showed good sensitivity, detecting Plasmodium and Babesia species with 100 % accuracy overall, except for P. falciparum (97.7 %) and B. microti (12.5 %). The low sensitivity of detecting B. microti was attributed to limitations in microscopy for species identification. This technique heavily relies on the proficiency of the examiner, as species within the genus cannot be distinguished under a microscope. Additionally, Babesia can be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. The findings support multiplex qPCR's diagnostic superiority over the gold standard, despite higher costs. It offers enhanced sensitivity, specificity, and detects mixed infections, crucial for effective monitoring and diagnosis of malaria and babesiosis in endemic regions with significant public health challenges.

Mapping the main harmful algal species in the East China Sea (Yangtze River estuary) and their possible response to the main ecological status and global climate change via a global vision.

The Yangtze River Estuary (YRE) is one of the areas in China most severely affected by harmful algal blooms (HABs). This study explored the distributive patterns of HABs in the YRE and how they are influenced by the El Niño-Southern Oscillation (ENSO) and other environmental factors. Quantitative real-time PCR (qPCR) was employed to detect and quantify the four predominant HAB species in the YRE, Karenia mikimotoi, Margalefidinium polykrikoides, Prorocentrum donghaiense, and Heterosigma akashiwo. Additionally, the study analyzed how turbidity, pH, salinity, and temperature influence these algae. Distribution of the four HAB species in the YRE area shows clear geographical variations: K. mikimotoi is predominantly found in the northwest and central sea areas, M. polykrikoides (East Asian Ribotype, EAR) is mainly distributed in the southeastern part, P. donghaiense is abundant in the northern regions, and H. akashiwo is especially prevalent at stations S26 and S27 in the northeastern part of the study area. HABs dominated by H. akashiwo and P. donghaiense were observed in the northeastern sea area of the YRE on July 22, 2020. Our study reveals that K. mikimotoi, M. polykrikoides (EAR), and P. donghaiense are mainly affected by turbidity, pH, and salinity, while temperature predominantly influences the blooms of H. akashiwo. Moreover, runoff in the YRE has a certain correlation with ENSO events, which may also impact the nutrient content of the region. The findings of this study illustrate the distributive patterns of the four HAB species under various ecological conditions in the YRE and emphasize the importance of establishing practical cases for future warning systems. To better understand how climate change affects HABs, exploring the link between ENSO and HABs is essential.

Impact of gastrointestinal inoculation and benznidazole treatment on infection by Trypanosoma cruzi (Y strain, DTU TcII) in Swiss mice.

In Brazil, where Chagas disease is endemic, the most frequent form of transmission of the parasite is the oral route, associated with greater severity and worse response to benznidazole (BZ), the drug used in its treatment. This study aimed to evaluate the impact of gastrointestinal infection (GI) and BZ treatment on the parasitological and histopathological parameters in mice inoculated with a strain of T. cruzi II. Swiss mice were inoculated by GI and intraperitoneal (IP) routes with 2x106 culture-derived metacyclic trypomastigotes of the Y strain (TcII) of T. cruzi and were treated with BZ in the acute phase of the infection. Fresh blood examination, qPCR, histopathological and biochemical evaluations (enzymatic dosages and oxidative stress-OS) were performed. BZ treatment of uninfected animals caused changes in the liver, increased the activity of aspartate aminotransferase and alanine aminotransferase enzymes and OS, showing that the drug alone affects this organ. Inflammation and necrosis in the cardiac tissue were less intense and deaths occurred later in animals inoculated via the GI route than the animals inoculated via the IP route. BZ reduced the intensity of tissue lesions and avoided lethality in animals inoculated via the GI route, and decreased parasitemia and OS in those inoculated via both routes. Although BZ alone caused liver damage, it was less intense than that caused by both routes of inoculation. Infection with the Y strain of T. cruzi II via the GI route proved to be less virulent and pathogenic and responded better to treatment than the infection acquired via the IP route.

Comparison of microbiological and molecular diagnosis for identification of respiratory secondary infections in COVID-19 patients and their antimicrobial resistance patterns.

We report the use of a new multiplex Real-Time PCR platform to simultaneously identify 24 pathogens and 3 antimicrobial-resistance genes directly from respiratory samples of COVID-19 patients. Results were compared to culture-based diagnosis. Secondary infections were detected in 60% of COVID-19 patients by molecular analysis and 73% by microbiological assays, with no significant differences in accuracy, indicating Gram-negative bacteria as the predominant species. Among fungal superinfections, Aspergillus spp. were detected by both methods in more than 7% of COVID-19 patients. Oxacillin-resistant S. aureus and carbapenem-resistant K. pneumoniae were highlighted by both methods. Secondary microbial infections in SARS-CoV-2 patients are associated with poor outcomes and an increased risk of death. Since PCR-based tests significantly reduce the turnaround time to 4 hours and 30 minutes (compared to 48 hours for microbial culture), we strongly support the routine use of molecular techniques, in conjunction with microbiological analysis, to identify co/secondary infections.

Analysis of chicken and pig DNA content in commercial dry foods for adult cats.

Among pets, cats are the most popular in Europe. Despite the fact, the interest in the safety and quality of their food is much lower compared to the interest of caregivers in the nutrition of dogs. In this research, 27 commercial cat foods were analyzed for mislabeled component composition. Cat foods were divided into a control group, a group of fish foods and a group of other foods with alternative sources of animal protein. Chicken and pig DNA detection was performed using real-time PCR. In this research, 100% of the cat foods contained chicken DNA and 96% of the foods - pig DNA, despite the lack of declaration of these ingredients on the product label. The results indicate that cat food appear to be mislabeled to an even greater extent than dog food. Moreover, manufacturers' declarations in terms of ingredient composition do not reflect the actual composition of commercial products available on the market and intended for everyday feeding of animals. Mislabeling of these products also poses a risk for animals suffering from food allergies.