Bacteria Culture Method Research Articles

Molecular Detection of Sapovirus in Children Under Five Years with Acute Gastroenteritis in Mansoura, Egypt between January 2019 and February 2020

Background: Sapovirus has emerged as a viral cause of acute gastroenteritis. However, there is limited data on sapovirus in Egypt. . The present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis <5 years in Mansoura, Egypt from January 2019 to February 2020 by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods: The cross-sectional study enrolled a 100 children <5 years who presented with acute gastroenteritis at an outpatient clinic in Mansoura, Egypt between January 2019 and February 2020. Clinical data, demographic data and a stool sample was collected from each child. Stools were screened by microscopy for parasites and culture methods for bacteria and excluded from the study if positive for either. Specimens were also screened for rotavirus by enzyme immune assays (EIA) and sapovirus by reverse transcription PCR. Results: The most frequently detected virus was rotavirus by ELISA 25% (25/100). RT-PCR detected sapovirus in 7% (7/100) of the stool samples. The children with sapovirus were all from rural regions and presented mainly during the winter season in Egypt 42.9% (3/7). The main presenting symptoms were fever 71.4% (5/7) and vomiting 57.1% (4/7). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in five samples (5/7) , 71.4%, P=0.01. Conclusion: The present study highlights the emergence of sapovirus as a frequent pathogen associated with acute gastroenteritis in children. There is a need for a national survey program for the study of sapovirus among other pathogens associated with acute gastroenteritis for better management of such infection.

Molecular Detection of Sapovirus in Children Under Five Years with Acute Gastroenteritis in Mansoura, Egypt between January 2019 and February 2020

Background: Sapovirus has emerged as a viral cause of acute gastroenteritis. However, there is limited data on sapovirus in Egypt. . The present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis <5 years in Mansoura, Egypt from January 2019 to February 2020 by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods: The cross-sectional study enrolled a 100 children <5 years who presented with acute gastroenteritis at an outpatient clinic in Mansoura, Egypt between January 2019 and February 2020. Clinical data, demographic data and a stool sample was collected from each child. Stools were screened by microscopy for parasites and culture methods for bacteria and excluded from the study if positive for either. Specimens were also screened for rotavirus by enzyme immune assays (EIA) and sapovirus by reverse transcription PCR. Results: The most frequently detected virus was rotavirus by ELISA 25% (25/100). RT-PCR detected sapovirus in 7% (7/100) of the stool samples. The children with sapovirus were all from rural regions and presented mainly during the winter season in Egypt 42.9% (3/7). The main presenting symptoms were fever 71.4% (5/7) and vomiting 57.1% (4/7). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in five samples (5/7) , 71.4%, P=0.01. Conclusion: The present study highlights the emergence of sapovirus as a frequent pathogen associated with acute gastroenteritis in children. There is a need for a national survey program for the study of sapovirus among other pathogens associated with acute gastroenteritis for better management of such infection.

Molecular Detection of Sapovirus in Children Under Five Years with Acute Gastroenteritis in Mansoura, Egypt between January 2019 and February 2020

Background: Sapovirus has emerged as a viral cause of acute gastroenteritis. However, there is limited data on sapovirus in Egypt. . The present study aimed to evaluate the presence of sapovirus in children with acute gastroenteritis <5 years in Mansoura, Egypt from January 2019 to February 2020 by reverse transcriptase-polymerase chain reaction (RT-PCR). Methods: The cross-sectional study enrolled a 100 children <5 years who presented with acute gastroenteritis at an outpatient clinic in Mansoura, Egypt between January 2019 and February 2020. Clinical data, demographic data and a stool sample was collected from each child. Stools were screened by microscopy for parasites and culture methods for bacteria and excluded from the study if positive for either. Specimens were also screened for rotavirus by enzyme immune assays (EIA) and sapovirus by reverse transcription PCR. Results: The most frequently detected virus was rotavirus by ELISA 25% (25/100). RT-PCR detected sapovirus in 7% (7/100) of the stool samples. The children with sapovirus were all from rural regions and presented mainly during the winter season in Egypt 42.9% (3/7). The main presenting symptoms were fever 71.4% (5/7) and vomiting 57.1% (4/7). None of the children with sapovirus had dehydration. Rotavirus was significantly associated with sapovirus infections in five samples (5/7) , 71.4%, P=0.01. Conclusion: The present study highlights the emergence of sapovirus as a frequent pathogen associated with acute gastroenteritis in children. There is a need for a national survey program for the study of sapovirus among other pathogens associated with acute gastroenteritis for better management of such infection.

Pathogen determination from clinical abscess fluids using metagenomic next-generation sequencing.

Abscesses are often clinically manifested as local necrotic tissues in various organs or systems of the human body, which is commonly caused by microbial infection. Rapid and accurate identification of pathogens from clinical abscetic samples would greatly guide a clinician to make the precise choices of the antimicrobial treatment. Here, this study aimed to investigate the application of metagenomic next-generation sequencing (mNGS) in the microbial detection of clinical samples of abscess fluids from various organs or systems. Nine patients with abscess from various organs or systems were enrolled in this study. The pathogenic bacteria in abscess fluid were detected and compared by the conventional bacterial culture and mNGS respectively. The dominant pathogens of abscess fluids in 8 cases can be found directly from mNGS, dominating over 80% of the total reads abundance of the microbiome. Although the pathogens from 6 cases detected by mNGS were consistent with that from the conventional bacteria culture method, the fastidious obligate anaerobic bacteria in 2 cases additionally detected by mNGS were not found by the conventional culture method. Moreover, complex polymicrobial infection containing Parvimonas micra in one case negatively with conventional bacterial culture were demonstrated by the mNGS method. And the mNGS method can directly reflect the diversity of microbial ecology in the abscess fluids from the different parts of the human body. Conclusively, mNGS can be used as a supplemental method for the pathogen detection of clinically abscess fluids.

Impact of donor lung colonized bacteria detected by next-generation sequencing on early post-transplant outcomes in lung transplant recipients

BackgroundThe effect of donor lung colonized bacteria on the prognosis of lung transplantation is not clear. We used the technique of next-generation sequencing (NGS) to detect the colonized bacteria from the lower respiratory tract and analyzed whether the colonized bacteria of donor lung could affect the outcomes of lung transplantation.MethodsSeventeen patients who underwent lung transplantation from March 2018 to June 2018 at Wuxi People’s Hospital affiliated to Nanjing Medical University were included in this study. Twelve cases of donor lung were obtained, and 17 lung transplants were performed, including 12 single lung transplantation and 5 bilateral lung transplantation. The colonized bacteria in the lower lobe tissue of donor lung were detected by NGS, and the bacteria culture method was used to detect the bacteria in the airway secretion before and after the operation. The information of length of extracorporeal membrane oxygenation (ECMO) support, mechanical ventilation time, length of intensive care unit (ICU) stay, duration of fever and length of hospital stay were collected for prognostic analysis.ResultsCompared with bacterial culture methods, the positive rate by using NGS in the lungs were higher (52.9% vs 41.2%). Among the patients who were transplanted with donor lungs with detected bacteria by NGS before surgery, only one patient (1/9) developed the same bacteria after lung transplantation. Based on results of NGS and bacterial culture, there was no association between the colonized bacteria in donor lungs and the patients’ outcomes of immediate posttransplant period.ConclusionNGS showed more sensitive than bacterial culture for detection of bacteria. The colonized bacteria in different parts of the lung are inconsistent. There is no association between the colonized bacteria in donor lungs and short-term outcome of lung transplantation patients.

Molecular detection of intrauterine microbial colonization in women with endometriosis

ObjectiveIncreased intrauterine microbial colonization by bacteria culture method and occurrence of endometritis have been reported in women with endometriosis. Here we investigated microbial colonization in intrauterine environment and cystic fluid of women with and without endometriosis by molecular approach. Study designThis is a case-controlled biological study with a total of 32 women each with and without endometriosis. Among them, 16 each in these two groups of women received treatment with gonadotropin-releasing hormone agonist (GnRHa). Pattern of microbial colonization in endometrial swabs and endometrioma/non-endometrioma cystic fluid was examined using broad-range polymerase-chain reaction (PCR) amplification of bacteria targeting 16S rRNA gene (rDNA). After quantification of index PCR product, 16S rDNA metagenome sequence analysis was done by Illumina Miseq system. ResultsA wide proportion (0.01–97.8%) of multiple bacteria was detected in both endometrial swabs and cystic fluid collected from women with and without endometriosis. 16S metagenome assay indicated that proportion of Lactobacillacae was significantly decreased (p<0.01) and of Streptococcaceae, Staphylococaceae, Enterobacteriaceae was significantly increased (p<0.05 for each) in GnRHa-treated women with endometriosis than in GnRHa-untreated women. While bacteria culture method failed to detect a single colony, 16S metagenome assay could detect significantly higher percentage of Streptococcaceae (p<0.01) and Staphylococaceae (p<0.05) in the cystic fluid derived from women with ovarian endometrioma comparing to that in cystic fluid collected from non-endometrioma cysts. ConclusionThese findings indicate the occurrence of sub-clinical infection in intrauterine environment and in the cystic fluid of ovarian endometrioma. Additional side effect of GnRHa treatment in promoting silent intrauterine and/or ovarian infection should be considered.

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

BackgroundPeptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.MethodsIdentification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.ResultsIn blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.ConclusionsIn this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.

Application of a Real-Time PCR Method for Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens Detection in Water Samples

The diagnostic assessment of water sanitary state is based mainly on the cultivation of bacteria retained on membrane filters. However classical microbiology methods have a lot of disadvantages. More and more frequently, rapid detection and identification of pathogens present in water is based on molecular biology techniques. The aim of this study was to determine the effectiveness and usefulness of a real-time PCR method, when compared to the recommended bacteria culture method, in diagnostics of pathogens in water samples. The research concerned the detection and identification of main sanitary indicators of water such as: Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens. The analyses were conducted in water samples contaminated with the reference material (the aforementioned bacteria) and real environmental samples, which were examined for the presence of nucleic acid of: Salmonella spp., E. coli, S. aureus and C. perfringens using a real-time PCR method.

Rapid parallelized and quantitative analysis of five pathogenic bacteria by ITS hybridization using QCM biosensor

We developed a 2 × 5 model quartz crystal microbalance (QCM) DNA biosensor array for detection of five bacteria, which based on hybridization analysis of bacterial 16S–23S rDNA internal transcribed spacer (ITS) region. A pair of universal primers was designed for PCR amplification of the ITSs. The PCR products were analyzed by the biosensor. We used gold nanoparticles to amplify the frequency shift signals. Fifty clinical samples were detected by both the biosensor and conventional bacteria culture method. We found a linear quantitative relationship between frequency shift and logarithmic concentration of synthesized oligonucleotides or bacteria cells. The measurable concentration ranged from 10 −12 to 10 −8 M for synthesized oligonucleotides and 1.5 × 10 2 to 1.5 × 10 8 CFU/mL for bacteria. The 10 −12 M of synthesized oligonucleotides or 1.5 × 10 2 CFU/mL of Pseudomonas aeruginosa could be detected by the biosensor system. The detection could be completed within 5 h including the PCR amplification procedure. Compared with bacteria culture method, the detection sensitivity and specificity of the biosensor system were 94.12% and 90.91%, respectively. There was no significant difference between these two methods ( P = 0.625 > 0.05). The biosensor system provides a rapid and sensitive method for parallelized and quantitative analysis of multiple pathogenic bacteria in clinical diagnosis.