Dataset Information

Molecular identification and genotyping of Acanthamoeba spp., in bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders (CRD).

ABSTRACT: This study aimed to investigate the presence and genotyping of Acanthamoeba spp., in the bronchoalveolar lavage fluid (BALF) of immunocompetent patients with chronic respiratory disorders (CRD). In this study, 211 BALF samples were collected from patients with CRD during the COVID-19 pandemic who were candidates for fiberoptic bronchoscopy (FOB) at Imam Khomeini Hospital, Sari, Mazandaran Province, northern Iran and investigated for Acanthamoeba spp., by PCR. A total of 211 FBAL samples were examined; 5 (5/211; 2.36%) were positive by using the PCR test for Acanthamoeba spp. According to sequence analysis, three strains belonged to the T4 genotype and one strain to the T2 genotype. Our data demonstrate that the presence of Acanthamoeba (T4 and T2) in BALF specimens of patients with respiratory infections. However, it is important to note that these findings may be merely accidental. Our findings suggest further investigation to fully understand the role of Acanthamoeba spp. in the pathogenesis of lung infections.

SUBMITTER: Saberi R

PROVIDER: S-EPMC9362591 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Molecular identification and genotyping of Acanthamoeba spp., in bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders (CRD).

Saberi Reza R Nakhaei Maryam M Fakhar Mahdi M Zarrinfar Hossein H Sharifpour Ali A Hezarjaribi Hajar Ziaei HZ

Parasitology research 20220805 10

This study aimed to investigate the presence and genotyping of Acanthamoeba spp., in the bronchoalveolar lavage fluid (BALF) of immunocompetent patients with chronic respiratory disorders (CRD). In this study, 211 BALF samples were collected from patients with CRD during the COVID-19 pandemic who were candidates for fiberoptic bronchoscopy (FOB) at Imam Khomeini Hospital, Sari, Mazandaran Province, northern Iran and investigated for Acanthamoeba spp., by PCR. A total of 211 FBAL samples were exa ...[more]

PMID: 35930044

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Project description:Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.

Project description:BackgroundIt is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data.ResultsUsing three mock communities across a range of densities ranging from 8E+ 02 to 8E+ 09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+ 05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+ 04 and 8E+ 06 16S copies/ml. For the lowest densities, below 8E+ 03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples.ConclusionRelative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+ 04 16S copies/ml.

Dataset Information

Molecular identification and genotyping of Acanthamoeba spp., in bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders (CRD).

Publications

Molecular identification and genotyping of Acanthamoeba spp., in bronchoalveolar lavage fluid from immunocompetent patients with chronic respiratory disorders (CRD).

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