scholarly journals Identification of the Bacterial Pathogens in Children with Otitis Media: A Study in the Northwestern Portuguese District of Braga

2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Maria Daniela Silva ◽  
António Lima ◽  
Nuno Marçal ◽  
Luís Dias ◽  
Miguel Gama ◽  
...  
Keyword(s):  
Otitis Media ◽  
Moraxella Catarrhalis ◽  
Pathogen Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Bacterial Identification ◽  
Chain Reaction ◽  
Middle Ear Fluid ◽  
Polymerase Chain ◽  
Polymicrobial Infections

Understanding the bacterial etiology of otitis media (OM) is important when designing and evaluating the best course of treatment. This study analyzed middle ear fluid (MEF) and nasopharynx (NP) samples collected from 49 children with OM undergoing myringotomy in the northwestern Portuguese district of Braga. A correlation between species in the NP and MEF was observed following pathogen detection by culture and quantitative polymerase chain reaction (qPCR) methods. Bacterial identification using culturing methods showed that Moraxella catarrhalis was the most representative in NP and MEF, followed by Streptococcus pneumoniae. However, qPCR of MEF showed a higher prevalence (61%) of Haemophilus influenzae. S. pneumoniae was not the most frequently identified species, but it still remains one of the leading causes of OM in this region despite 93.9% of the children being vaccinated with the pneumococcal conjugate vaccine. Furthermore, 46% of the samples analyzed by qPCR identified more than two bacterial species. M. catarrhalis and S. pneumoniae were the most frequent combination identified in NP and MEF samples by culturing methods. Additionally, a few NP and MEF samples simultaneously presented the three main otopathogens. These results point out that polymicrobial infections play an important role in OM. Further studies characterizing the serotypes of the strains isolated, their resistance profile, and their biofilm forming ability would help in the development of more targeted strategies against otitis media.

scholarly journals Airway Bacteria Quantification Using Polymerase Chain Reaction Combined with Neutrophil and Eosinophil Counts Identifies Distinct COPD Endotypes

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1337
Author(s):  
Augusta Beech ◽  
Simon Lea ◽  
Jian Li ◽  
Natalie Jackson ◽  
Alex Mulvanny ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Bacterial Colonisation ◽  
Chain Reaction ◽  
Cell Counts ◽  
Copd Patients ◽  
Polymerase Chain ◽  
Eosinophil Counts

Background: Chronic obstructive pulmonary disease (COPD) inflammatory endotypes are associated with different airway microbiomes. We used quantitative polymerase chain reaction (qPCR) analysis of sputum samples to establish the bacterial load upper limit in healthy controls; these values determined the bacterial colonisation prevalence in a longitudinal COPD cohort. Bacteriology combined with sputum inflammatory cells counts were used to investigate COPD endotypes. Methods: Sixty COPD patients and 15 healthy non-smoking controls were recruited. Sputum was analysed by qPCR (for Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Psuedomonas aeruginosa) and sputum differential cell counts at baseline and 6 months. Results: At baseline and 6 months, 23.1% and 25.6% of COPD patients were colonised with H. influenzae, while colonisation with other bacterial species was less common, e.g., S. pneumoniae—1.9% and 5.1%, respectively. H. influenzae + ve patients had higher neutrophil counts at baseline (90.1% vs. 67.3%, p < 0.01), with similar results at 6 months. COPD patients with sputum eosinophil counts ≥3% at ≥1 visit rarely showed bacterial colonisation. Conclusions: The prevalence of H. influenzae colonisation was approximately 25%, with low colonisation for other bacterial species. H. influenzae colonisation was associated with sputum neutrophilia, while eosinophilic inflammation and H. influenzae colonisation rarely coexisted.

scholarly journals Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius)

2015 ◽  
Vol 9 (10) ◽  
pp. 1117-1125 ◽  
Author(s):  
Hanène Belkahia ◽  
Mourad Ben Said ◽  
Lotfi Sayahi ◽  
Alberto Alberti ◽  
Lilia Messadi
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Chain Reaction ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Nested Polymerase Chain Reaction ◽  
16S Rrna Sequence Analysis ◽  
Polymerase Chain

Introduction: Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. Methodology: A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. Results: Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. Conclusions: This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.

scholarly journals Performance of TEM-PCR vs Culture for Bacterial Identification in Pediatric Musculoskeletal Infections

2018 ◽  
Vol 5 (6) ◽  
Author(s):  
James B Wood ◽  
Cheryl Sesler ◽  
Donald Stalons ◽  
Elena Grigorenko ◽  
Jonathan G Schoenecker ◽  
...  
Keyword(s):  
Multiplex Polymerase Chain Reaction ◽  
Pathogen Detection ◽  
Susceptibility Testing ◽  
Antimicrobial Use ◽  
Antibiotic Susceptibility Testing ◽  
Disease Course ◽  
Bacterial Identification ◽  
Pathogen Identification ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Improved diagnostics are needed for children with musculoskeletal infections (MSKIs). We assessed the performance of target-enriched multiplex polymerase chain reaction (TEM-PCR) in children with MSKI. TEM-PCR was concordant with culture in pathogen identification and antibiotic susceptibility testing, while increasing the overall yield of pathogen detection. This technology has the potential to inform judicious antimicrobial use early in the disease course.

Fungal Diversity and Onychomycosis

2019 ◽  
Vol 109 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Annette Joyce ◽  
Aditya K. Gupta ◽  
Lars Koenig ◽  
Randall Wolcott ◽  
Jessie Carviel
Keyword(s):  
Fungal Diversity ◽  
Potassium Hydroxide ◽  
Trichophyton Rubrum ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
The United States ◽  
Molecular Methods ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Generation Sequencing

Background: Onychomycosis is a fungal infection of the nail that is often recalcitrant to treatment and prone to relapse. Traditional potassium hydroxide and culture diagnosis is costly and time-consuming. Therefore, molecular methods were investigated to demonstrate effectiveness in diagnosis and to quantify the microbial flora present that may be contributing to disease. Methods: A total of 8,816 clinically suspicious toenail samples were collected by podiatric physicians across the United States from patients aged 0 to 103 years and compared with a control population (N = 20). Next-generation sequencing and quantitative polymerase chain reaction were used to identify and quantify dermatophytes, nondermatophyte molds, and bacteria. Results: Approximately 50% of suspicious toenails contained both fungi and bacteria, with the dermatophyte Trichophyton rubrum contributing the highest relative abundance and presence in 40% of these samples. Of the remaining 50% of samples, 34% had bacterial species present and 16% had neither. Fungi only were present in less than 1% of samples. Nondermatophyte molds contributed to 11.0% of occurrences in fungus-positive samples. All of the control samples were negative for fungi, with commensal bacterial species composing most of the flora population. Conclusions: Molecular methods were successful in efficiently quantifying microbial and mycologic presence in the nail. Contributions from dermatophytes were lower than expected, whereas the opposite was true for nondermatophyte molds. The clinical significance of these results is currently unknown.

scholarly journals Validating Quantitative Polymerase Chain Reaction Assay for the Molecular Diagnosis of Chronic Suppurative Otitis Media

2020 ◽  
Vol 8 (A) ◽  
pp. 491-497
Author(s):  
Dina Alia ◽  
Ferry Dwi Kurniawan ◽  
Azwar Ridwan ◽  
Wilda Mahdani ◽  
Koichi Hagiwara
Keyword(s):  
Polymerase Chain Reaction ◽  
Otitis Media ◽  
Bacterial Culture ◽  
Quantitative Polymerase Chain Reaction ◽  
Chronic Suppurative Otitis Media ◽  
Chain Reaction ◽  
Suppurative Otitis Media ◽  
Receiver Operating Characteristic Curves ◽  
Cycle Threshold ◽  
Polymerase Chain

BACKGROUND: Pathogen identification is critical for antibiotic selection in suppurative otitis media. However, bacterial culture challenges from suppurative specimens often cause antibiotic misuse and ineffective treatment. A quantitative polymerase chain reaction (PCR) controlled by the human cells contained in the specimen (HIRA-TAN) has been established in differentiate between pathogens and colonization in the previous pneumonia study. AIM: The aim of this study was to investigate the utility of HIRA-TAN and determine the causative pathogen in chronic suppurative otitis media. METHODS: Thirty-nine patients were recruited to the study. The otorrhea was swab-collected and processed for both bacterial culture and a multiplex PCR-based test. The cutoff of cycle threshold to determinate the pathogens was defined by receiver operating characteristic curves. RESULTS: Thirty-nine patients ranging from 1.7 to 62 years old were enrolled. The hearing impairment was found different between adult and children (p < 0.005) with adults (24/29 patients) had a significantly higher rate. A total of 35.9% of samples were positive for bacterial culture; Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Morganella morganii, while Bacteroides fragilis, Acinetobacter baumannii, Moraxella catarrhalis, and Escherichia coli were not identified by culture, although high cycle-threshold values were obtained suggesting the inability of the culture system in detecting some pathogens. CONCLUSION: Our results indicate that HIRA-TAN is a potential diagnostic tool in suppurative otitis media and warrant a larger cohort study.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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