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Microorganisms Jun 2024We present the validity of using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for quantifying high-risk human papillomavirus (HPV) 16 E7 oncoproteins in...
We present the validity of using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) for quantifying high-risk human papillomavirus (HPV) 16 E7 oncoproteins in urine specimens as a noninvasive method of analyzing the oncogenic activity of HPV. Some reports claim that the oncogenic activity of HPV is a more relevant clinical indicator than the presence of HPV DNA for estimating malignant potential. In the present study, urine containing HPV16 and related types were selected by uniplex E6/E7 polymerase chain reaction and classified according to the pathologic diagnosis of cervical intraepithelial neoplasia (CIN) in cervical biopsy specimens. Our ultrasensitive ELISA was able to detect attomole levels of HPV16 E7 oncoproteins, and it detected HPV16-positive SiHa cells at >500 cells/mL without detecting HPV18-positive cells. Our ELISA results showed E7 oncoproteins in 80% (4/5) of urine specimens from women with HPV16-positive CIN1, 71% (5/7) of urine specimens from CIN2 patients, and 38% (3/8) of urine specimens from CIN3 patients. Some urine specimens with undetectable E7 oncoproteins were thought to be negative for live HPV 16-positive cells or in an inactivated state of infection. These results provide the basis for assessing oncogenic activity by quantifying E7 oncoproteins in patient urine.
PubMed: 38930587
DOI: 10.3390/microorganisms12061205 -
Microorganisms Jun 2024In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of . Observations of fresh mounts revealed...
In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of . Observations of fresh mounts revealed structures resembling lipid droplets, similar to those seen in a condition known as "las bolitas syndrome". Routine histopathological examinations identified detached cells and tissues in the digestive tracts of affected zoea, contrasting with the typical algal cell contents seen in healthy zoea. Polymerase chain reaction (PCR) testing for over 20 known shrimp pathogens indicated minimal differences between diseased and healthy batches. Both groups tested negative for acute hepatopancreatic necrosis disease (AHPND) but positive for species and Rickettsia-like bacteria in the diseased samples. Histological analyses of the affected zoea revealed characteristic tissue degeneration in the hepatopancreas, forming spheres that eventually migrated into the upper gut, midgut, and midgut caeca, a pathology identified as bolitas syndrome (BS). Microbiological assessments revealed species at concentrations of 10 CFU zoea/g in affected zoea, approximately two orders of magnitude higher than in healthy zoea. Bacterial isolation from both healthy and BS-affected zoea on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and CHROMagar™ (Paris, France), followed by identification using API 20E, identified six strains of . Despite similarities to "las bolitas syndrome" in fresh mounts, distinct histopathological differences were noted, particularly the presence of sloughed cells in the intestines and variations in hepatopancreatic lobes. This study highlights the critical need for further research to fully understand the etiology and pathology of bolitas syndrome in zoea stage 2 of to develop effective mitigation strategies for hatchery operations.
PubMed: 38930568
DOI: 10.3390/microorganisms12061186 -
Microorganisms Jun 2024Better diagnostic tools are needed to improve the diagnosis of infections (CDI) and reduce the overtreatment of colonized children. In this study, we evaluated two...
Better diagnostic tools are needed to improve the diagnosis of infections (CDI) and reduce the overtreatment of colonized children. In this study, we evaluated two polymerase chain reaction (PCR) assays (Cepheid GeneXpert and the Gastroenteritis PCR Panel by QIAstat-Dx) as a standalone method in combination with the PCR cycle threshold (Ct) value in positive samples to predict the presence of free toxins. We also evaluated the clinical impact of reporting toxin production results and provided comments alongside the PCR results in our pediatric population. PCR-positive stool samples from pediatric patients (aged 2 to 18 years old) were included in our study and tested for the presence of toxins A and B using the Quik Chek Complete kit. For the clinical intervention, the CDI treatment rates 6 months pre- and post-intervention were compared. The use of PCR Ct value showed excellent sensitivity (100%) at a Ct value cutoff of 26.1 and 27.2 using the Cepheid GeneXpert and the Gastroenteritis PCR Panel by QIAstat-Dx, respectively, while the toxin test showed inferior sensitivity of 64% in the PCR-positive samples. In addition, CDI treatment rates were decreased by 23% post-intervention. The results of our study suggest that nucleic acid amplification test (NAAT) assays supplemented by the use of PCR Ct value for positive samples can be used as standalone tests to differentiate CDI from colonization. Furthermore, the reporting of toxin production along with the PCR results can help reduce the unnecessary treatment of colonized children.
PubMed: 38930564
DOI: 10.3390/microorganisms12061181 -
Microorganisms Jun 2024Salt-tolerant aerobic granular sludge(AGS) was successfully cultivated under the dual stress of tetracycline and 2.5% salinity, resulting in an average particle size of...
Salt-tolerant aerobic granular sludge(AGS) was successfully cultivated under the dual stress of tetracycline and 2.5% salinity, resulting in an average particle size of 435.0 ± 0.5 and exhibiting a chemical oxygen demand(COD) removal rate exceeding 80%, as well as excellent sedimentation performance. The analysis of metagenomics technology revealed a significant pattern of succession in the development of AGS. The proportion of , a type of salt-tolerant bacteria, exhibited a gradual increase and reached 38.07% after 42 days, which indicated that an AGS system based on moderate halophilic bacteria was successfully constructed. The expression levels of targeted genes were found to be reduced across the entire AGS process and formation, as evidenced by qPCR analysis. The presence of (7.67 log10 gene copies g in 0 d sludge sample) enabled microbes to horizontally transfer ARGs genes along the AGS formation under the double pressure of TC and 2.5% salinity. These findings will enhance our understanding of ARG profiles and the development in AGS under tetracycline pressure, providing a foundation for guiding the use of AGS to treat hypersaline pharmaceutical wastewater.
PubMed: 38930555
DOI: 10.3390/microorganisms12061173 -
Microorganisms Jun 2024The Esx-1 family proteins of the Type VII secretion systems of and have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both...
The Presence of and and Other Gene Orthologs of the RD 1 Region in Non-Tuberculous Mycobacteria, Mycolicibacteria, Mycobacteroides and Mycolicibacter as Possible Impediments for the Diagnosis of (Animal) Tuberculosis.
The Esx-1 family proteins of the Type VII secretion systems of and have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both humans and animals. The presence of ESAT-6 and CFP 10 proteins, which are the most immunogenic proteins of the Esx-1 system and have been widely investigated for the immunodiagnosis of tuberculosis, in some and in , poses limitations for their use in specific diagnoses of TB. As such, to improve the specificity of the ESAT-6/CFP 10-based cell-mediated immunity (CMI) assays, other proteins encoded by genes within and outside the RD 1 region of the esx-1 locus have been evaluated as candidate antigens for CMI, as well as to investigate humoral responses in combination with ESAT-6 and or CFP 10, with varying specificity and sensitivity results. Hence, in this study, we evaluated various non-tuberculous mycobacteria (NTM), , and species genomes available on the NCBI database for the presence and composition of the RD1 region of the esx-1 locus. In addition, we also assayed by polymerase chain reaction (PCR) and sequencing of available in our culture collection for the presence and sequence diversity of and genes encoding ESAT-6 and CFP 10, respectively. Whole genome sequence (WGS) data analysis revealed the presence of RD 1 gene orthologs in 70 of the over 100 published genomes of pathogenic and non- pathogenic other than tuberculosis. Among species evaluated from our culture collection, in addition to earlier reports of the presence of and in certain , , and sp. N845T were also found to harbour orthologs of both genes. Orthologs of only were detected in , and , whereas in , and , only orthologs were detected. A phylogenetic analysis based on and sequences separated slow-growing from rapidly growing bacteria. These findings strengthen previous suggestions that and may be encoded in the majority of . The role of the Esx-1 system in both pathogenic and non-pathogenic needs further investigation, as these species may pose limitations to immunological assays for TB.
PubMed: 38930534
DOI: 10.3390/microorganisms12061151 -
Microorganisms Jun 2024In the present study, we compared the genetic variability of fragments from the internal transcribed spacer region (ITS) and the small subunit ribosomal DNA (SSUrDNA) as...
Genetic Variation among the Partial Gene Sequences of the Ribosomal Protein Large-Two, the Internal Transcribed Spacer, and the Small Ribosomal Subunit of sp. from Human Fecal Samples.
In the present study, we compared the genetic variability of fragments from the internal transcribed spacer region (ITS) and the small subunit ribosomal DNA (SSUrDNA) as nuclear markers, in contrast with the ribosomal protein large two () , placed in the mitochondrion-related organelles (MROs) within and among human fecal samples with . Samples were analyzed using polymerase chain reaction (PCR)-sequencing, phylogenies, and genetics of population structure analyses were performed. In total, 96 sequences were analyzed, i.e., 33 of SSUrDNA, 35 of , and 28 of ITS. Only three subtypes (STs) were identified, i.e., ST1 (11.4%), ST2 (28.6%), and ST3 (60%); in all cases, kappa indexes were 1, meaning a perfect agreement among ST assignations. The topologies of phylogenetic inferences were similar among them, clustering to each ST in its specific cluster; discrepancies between phylogeny and assignment of STs were not observed. The STRUCTURE v2.3.4 software assigned three subpopulations corresponding to the STs 1-3, respectively. The population indices were consistent with those previously reported by other groups. Our results suggest the potential use of the ITS and genes as molecular markers for subtyping as an alternative approach for the study of the genetic diversity observed within and between human isolates of this microorganism.
PubMed: 38930533
DOI: 10.3390/microorganisms12061152 -
Microorganisms May 2024Leptospirosis is an infectious disease that affects domestic animals, wild animals, and humans. It represents a public health problem and has an important economic...
Leptospirosis is an infectious disease that affects domestic animals, wild animals, and humans. It represents a public health problem and has an important economic impact on livestock. This study aims to investigate the importance of genital and transplacental infection in the epidemiology of leptospirosis in cows maintained in Caatinga biome conditions, Northeastern Brazil, as well as reporting organs colonized by spp. in embryos and fetuses. Blood, urinary tract (urine, bladder, and kidney), and reproductive tract (vaginal fluid, uterus, uterine tube, ovary, and placenta) samples were collected from 15 slaughtered pregnant cows. Two embryos and 13 fetuses were sampled. Central nervous system and choroid ovoid samples were collected from embryos. Blood, central nervous system, lung, peritoneal liquid, abomasal content, liver, spleen, urine, bladder, kidney, and reproductive system samples were collected from fetuses. Diagnostic methods included the microscopic agglutination test (MAT) using a collection of 24 serovars belonging to 17 different pathogenic serogroups of five species as antigens, as well as polymerase chain reaction (PCR). Anti- spp. antibodies were found in 9 cows (60%), while 13 cows (86.67%) had at least one organ or urine with leptospiral DNA. No fetus was seroreactive. Among the embryos and fetuses, 13 (86.67%) presented leptospiral DNA, proving a high frequency of transplacental infection (100%). For cows, the most frequent biological materials regarding spp. DNA detection were placenta (13 out of 15 samples; 86.7%), uterus (10 out of 15 samples; 66.7%), and vaginal fluid (5 out of 15 samples; 33.3%), while, for fetuses/embryos, the most frequent PCR-positive samples were choroid ovoid (1/2; 50%), spleen (6/13; 46.2%), kidney (5/13; 38.5%), and central nervous system (5/15; 33.3%). Sequenced samples based on the LipL32 gene presented 99% similarity with . The results indicate that transplacental infection is an efficient way of spreading spp. in cows maintained in Caatinga biome conditions. Therefore, prevention and control strategies must include actions that interrupt transmission through this alternative route.
PubMed: 38930426
DOI: 10.3390/microorganisms12061044 -
Life (Basel, Switzerland) Jun 2024The genus holds economic significance due to its widespread distribution and diverse applications, including biological control, enzyme production, and various...
The genus holds economic significance due to its widespread distribution and diverse applications, including biological control, enzyme production, and various biotechnological uses. The accurate identification of species is crucial given their close association with human activities. Despite previous efforts in classification, a comprehensive analysis combining morphological and molecular approaches is necessary. This study focuses on the isolation of four species from industrial wastewater in Pakistan, expanding on the known diversity in the region; isolation involved collecting samples from industrial wastewater effluents at specific sites in Punjab, Pakistan. strains were cultured and purified on solid media, with subsequent biomass production for bisorptional activity. Morphological characterization included colony features and microscopic examinations. DNA extraction, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer (ITS) region were conducted for molecular analysis. Phylogenetic analysis was performed using the Maximum Likelihood Algorithm. The study identified three species, viz. , , and . Each species was characterized morphologically and supported by molecular-phylogenetic analysis. Illustrations of microscopic features and a phylogenetic tree based on the ITS-nrDNA region were recorded. and , isolated from steel mill and tanneries wastewater, respectively, were differentiated based on morphological characteristics such as phialides and conidia. The combination of morphological and molecular techniques enhances the accuracy of species identification. The study highlights the significance of in industrial wastewater environments and underscores the need for continued research in this area. Future research should focus on exploring the ecological roles and potential applications of the newly identified species. Additionally, further investigations into the biotechnological potential of these species, including enzyme production and bioremediation capabilities, would contribute to their practical applications.
PubMed: 38929733
DOI: 10.3390/life14060750 -
Medicina (Kaunas, Lithuania) Jun 2024: Malaria continues to be a significant global health challenge. The efficacy of artemisinin-based combination therapies (ACTs) has declined in many parts of the Greater...
: Malaria continues to be a significant global health challenge. The efficacy of artemisinin-based combination therapies (ACTs) has declined in many parts of the Greater Mekong Subregion, including Vietnam, due to the spread of resistant malaria strains. This study was conducted to assess the efficacy of the Dihydroartemisinin (DHA)-Piperaquine (PPQ) regimen in treating uncomplicated malaria and to conduct molecular surveillance of antimalarial drug resistance in Binh Phuoc and Dak Nong provinces. : The study included 63 uncomplicated malaria falciparum patients from therapeutic efficacy studies (TES) treated following the WHO treatment guidelines (2009). Molecular marker analysis was performed on all 63 patients. Methods encompassed Sanger sequencing for mutations and quantitative real-time PCR for the gene. : This study found a marked decrease in the efficacy of the DHA-PPQ regimen, with an increased rate of treatment failures at two study sites. Genetic analysis revealed a significant presence of mutations and amplifications, indicating emerging resistance to artemisinin and its partner drug. : The effectiveness of the standard DHA-PPQ regimen has sharply declined, with rising treatment failure rates. This decline necessitates a review and possible revision of national malaria treatment guidelines. Importantly, molecular monitoring and clinical efficacy assessments together provide a robust framework for understanding and addressing detection drug resistance in malaria.
Topics: Humans; Artemisinins; Quinolines; Vietnam; Antimalarials; Malaria, Falciparum; Male; Female; Adult; Plasmodium falciparum; Drug Resistance; Adolescent; Middle Aged; Drug Therapy, Combination; Young Adult; Protozoan Proteins; Real-Time Polymerase Chain Reaction; Mutation; Piperazines
PubMed: 38929629
DOI: 10.3390/medicina60061013 -
Medicina (Kaunas, Lithuania) May 2024: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and ranges from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and...
: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and ranges from simple steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Accumulating evidence in animal models suggests that loss of interleukin-10 (IL-10) anti-inflammatory actions might contribute to lobular inflammation, considered one of the first steps toward NASH development. However, the role of IL-10 in lobular inflammation remains poorly explored in humans. We examined mRNA and protein levels of IL-10 in liver biopsies and serum samples from morbidly obese patients, investigating the relationship between IL-10 and lobular inflammation degree. : We prospectively enrolled morbidly obese patients of both sexes, assessing the lobular inflammation grade by the Brunt scoring system to categorize participants into mild ( = 7), moderate ( = 19), or severe ( = 13) lobular inflammation groups. We quantified the hepatic mRNA expression of IL-10 by quantitative polymerase chain reaction and protein IL-10 levels in liver and serum samples by Luminex Assay. We estimated statistical differences by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. : The hepatic expression of IL-10 significantly diminished in patients with severe lobular inflammation compared with the moderate lobular inflammation group ( = 0.01). The hepatic IL-10 protein levels decreased in patients with moderate or severe lobular inflammation compared with the mild lobular inflammation group ( = 0.008 and = 0.0008, respectively). In circulation, IL-10 also significantly decreased in subjects with moderate or severe lobular inflammation compared with the mild lobular inflammation group ( = 0.005 and < 0.0001, respectively). : In liver biopsies and serum samples of morbidly obese patients, the protein levels of IL-10 progressively decrease as lobular inflammation increases, supporting the hypothesis that lobular inflammation develops because of the loss of the IL-10-mediated anti-inflammatory counterbalance.
Topics: Humans; Interleukin-10; Obesity, Morbid; Female; Male; Adult; Middle Aged; Liver; Prospective Studies; Inflammation; Non-alcoholic Fatty Liver Disease
PubMed: 38929479
DOI: 10.3390/medicina60060862