Helicobacter pylori is a bacterium that is present in the stomach of about 50% of the global population and is associated with several gastric disorders, including cancer. Natural products with antimicrobial activity have been tested against H. pylori, among them Trichilia catigua (catuaba), which is widely distributed in Brazil. This study aimed to evaluate extracts of T. catigua bark against H. pylori via determination of the minimum inhibitory and bactericidal concentrations (MIC and MBC); evaluation of virulence factors by real-time PCR, synergism with standard antimicrobials and morphology by scanning electron microscopy and simulations of the mechanism of action by molecular docking. The ethyl acetate fraction provided the best results, with an MIC50 of 250 μg/mL and a 42.34% reduction in urease activity, along with reduced expression of the CagA and VacA genes, which encode for the main virulence factors. This fraction presented synergistic activity with clarithromycin, reducing the MIC of the drug by four-fold. Docking simulations suggested that the extracts inhibit fatty acid synthesis by the FAS-II system, causing damage to the cell membrane. Therefore, T. catigua extracts have potential as an adjuvant to treatment and are promising for the development of new anti-H. pylori drugs.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA.
METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA).
RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12.
CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.

Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

Several single nucleotide polymorphisms (SNPs) in multiple interleukin receptor genes could be associated with asthma risk and/or phenotype. Interleukin-17 (IL-17) has been implicated in tissue inflammation and autoimmune diseases. As no previous studies have uncovered the potential role of IL17 receptor A (RA) gene variants in asthma risk, we aimed to explore the association of four IL17RA SNPs (i.e., rs4819554A/G, rs879577C/T, rs41323645G/A, and rs4819555C/T) with asthma susceptibility/phenotype in our region. TaqMan allelic discrimination analysis was used to genotype 192 individuals. We found that the rs4819554 G/G genotype significantly reduced disease risk in the codominant (OR = 0.15, 95%CI = 0.05-0.45, p < 0.001), dominant (OR = 0.49, 95%CI = 0.26-0.93, p = 0.028), and recessive (OR = 0.18, 95%CI = 0.07-0.52, p < 0.001) models. Similarly, rs879577 showed reduced disease risk associated with the T allele across all genetic models. However, the A allele of rs41323645 was associated with increased disease risk in all models. The G/A and A/A genotypes have higher ORs of 2.47 (95%CI = 1.19-5.14) and 3.86 (95%CI = 1.62-9.18), respectively. Similar trends are observed in the dominant 2.89 (95%CI = 1.47-5.68, p = 0.002) and recessive 2.34 (95%CI = 1.10-4.98, p = 0.025) models. For the rs4819555 variant, although there was no significant association identified under any models, carriers of the rs4819554*A demonstrated an association with a positive family history of asthma (71.4% in carriers vs. 27% in non-carriers; p = 0.025) and the use of relievers for >2 weeks (52.2% of carriers vs. 28.8% of non-carriers; p = 0.047). Meanwhile, the rs4819555*C carriers displayed a significant divergence in the asthma phenotype, specifically atopic asthma (83.3% vs. 61.1%; p = 0.007), showed a higher prevalence of chest tightness (88.9% vs. 61.5%; p = 0.029), and were more likely to report comorbidities (57.7% vs. 16.7%, p = 0.003). The most frequent haplotype in the asthma group was ACAC, with a frequency of 22.87% vs. 1.36% in the controls (p < 0.001). In conclusion, the studied IL17RA variants could be essential in asthma susceptibility and phenotype in children and adolescents.

BACKGROUND: Detecting Helicobacter pylori in fecal samples is easier and more comfortable than invasive techniques, especially in children. Thus, the objective of the present work was to detect H. pylori in feces from children by molecular methods as an alternative for diagnostic and epidemiological studies.
METHODS: Forty-five fecal samples were taken from pediatric patients who presented symptoms compatible with H. pylori infection. HpSA test, culture, real-time quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), direct viable count associated with FISH (DVC-FISH), and Illumina-based deep-amplicon sequencing (DAS) were applied.
RESULTS: No H. pylori colonies were isolated from the samples. qPCR analysis detected H. pylori in the feces of 24.4% of the patients. In comparison, DVC-FISH analysis showed the presence of viable H. pylori cells in 53.3% of the samples, 37% of which carried 23S rRNA mutations that confer resistance to clarithromycin. After DAS, H. pylori-specific 16S rDNA sequences were detected in 26 samples. In addition, DNA from H. hepaticus was identified in 10 samples, and H. pullorum DNA was detected in one sample.
CONCLUSIONS: The results of this study show the presence of H. pylori, H. hepaticus, and H. pullorum in children\'s stools, demonstrating the coexistence of more than one Helicobacter species in the same patient. The DVC-FISH method showed the presence of viable, potentially infective H. pylori cells in a high percentage of the children\'s stools. These results support the idea that fecal-oral transmission is probably a common route for H. pylori and suggest possible fecal-oral transmission of other pathogenic Helicobacter species.

Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. SHOX2 gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting SHOX2 methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated SHOX2 DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated SHOX2 DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting SHOX2 methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.

The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson\'s r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.

Porcine cytomegalovirus (PCMV) infection is widespread worldwide and has a high prevalence in swine herds, especially in countries with intensive swine production. PCMV is zoonotic and can impact xenotransplants. It is the third swine virus known to be zoonotic, following swine influenza virus (influenza A) and hepatitis E virus genotype 3 (HEVgt3 or HEV-3). Wild boars, serving as reservoirs for various pathogens, including PCMV, pose a risk to both the pig industry and public health. This study aimed to investigate PCMV infection in Serbian wild boars using real-time PCR and assess other viral infections. We also tested samples for the presence of other viral infections: Aujeszky disease virus (ADV), Porcine parvovirus (PPV) and Porcine reproductive respiratory syndrome (PRRSV). Samples from 50 wild boars across 3 districts were tested. Results showed 8% positivity for PCMV DNA, with females showing higher infection rates. Porcine parvovirus (PPV) was detected in 56% of samples, while Porcine reproductive respiratory syndrome virus (PRRSV) was absent. ADV was found in 18% of samples, primarily in younger animals. This research contributes to understanding PCMV prevalence in Serbian wild boars and emphasizes the importance of monitoring viral infections in wild populations, considering the potential zoonotic and economic implications.

Periodontal diseases, as an important part of oral pathology, present different characteristics when affecting children and adolescents or young adults. Studies have shown that adolescence and childhood are closely related to a high risk of periodontal disease, but the follow-up for periodontal health or damage at this age has been insufficiently appreciated until now. The aim of this study was to identify subgingival microorganisms using a real-time polymerase chain reaction (PCR) in a group of children and adolescents aged 7-17 years with and without cardiovascular disease. The group of 62 subjects with gingival inflammation and poor hygiene was divided into two groups according to general condition: 31 subjects with carduivascular disease (group A) and 31 subjects without cardiovascular disease (group C). Subjects were examined in the initial consultation, the state of hygiene and periodontal inflammation was assessed using the plaque index (PI) and gingival index (GI), and samples were taken from the gingival sulcus using sterile paper cones to determine nine subgingival microorganisms. Nine subgingival microorganisms were identified: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythias (Tf), Prevotella intermedia (Pi), Peptostreptococcus (Micromonas) micros (Pm), Fusobacterium nucleatum (Fn), Eubacterium nodatum (En), and Capnocytophaga gingivalis (Cg). The patients were included in a specialist treatment program which aimed to relieve the inflammatory condition, remove local irritative factors, and train the patients to perform proper oral hygiene at home by using primary and secondary oral hygiene products. Subjects were reevaluated 3 months after treatment, when measurements for the PI and GI and microbiological determinations were repeated. The results showed a predominance of subjects aged 16-17 years (12.4%). Among the subjects with marked gingival inflammation, the male gender was predominant (58.06%). The PI values changed considerably after treatment, with lower values in patients presenting a general condition without cardiovascular disease (PI = 8.10%) compared with the patients with cardiovascular disease (PI = 13.77%). After treatment, the GI showed considerable changes in both groups. Red, orange, and purple complex microorganisms were found before treatment and decreased considerably after treatment in both groups. The highest values were found for Treponema denticola (140,000 (1.4 × 105)) in patients with cardiovascular disease and generalized gingival inflammation. Of the pathogenic microorganisms, the most common was Tannerella forsythia in 52 patients before treatment, and red microorganisms considerably appeared in only 10 patients after treatment. Capnocytophaga gingivalis remained constant both in the diseased state and after treatment and was consistent with periodontal health. Children with cardiovascular diseases had a higher prevalence of gingival manifestations. The composition of the subgingival microbial plaque was directly influenced by the degree of oral hygiene, but the response to specialized treatment was also influenced by the general health status. The results of this study support the conclusion that periodontal pathogens appear and multiply in the absence of proper hygiene in childhood after the eruption of permanent teeth, and their action leads to the initiation of periodontal diseases.