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1.
Micromachines (Basel) ; 12(12)2021 Dec 19.
Article in English | MEDLINE | ID: covidwho-1580576

ABSTRACT

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 µL and 10.6 µL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

2.
BMC Microbiol ; 21(1): 277, 2021 10 11.
Article in English | MEDLINE | ID: covidwho-1463230

ABSTRACT

BACKGROUND: Fusobacterium nucleatum (F. n) is an important opportunistic pathogen causing oral and gastrointestinal disease. Faecalibacterium prausnitzii (F. p) is a next-generation probiotic and could serve as a biomarker of gut eubiosis/dysbiosis to some extent. Alterations in the human oral and gut microbiomes are associated with viral respiratory infection. The aim of this study was to characterise the oral and fecal bacterial biomarker (i.e., F. n and F. p) in COVID-19 patients by qPCR and investigate the pharyngeal microbiome of COVID-19 patients through metagenomic next-generation sequencing (mNGS). RESULTS: Pharyngeal F. n was significantly increased in COVID-19 patients, and it was higher in male than female patients. Increased abundance of pharyngeal F. n was associated with a higher risk of a positive SARS-CoV-2 test (adjusted OR = 1.32, 95% CI = 1.06 ~ 1.65, P < 0.05). A classifier to distinguish COVID-19 patients from the healthy controls based on the pharyngeal F. n was constructed and achieved an area under the curve (AUC) of 0.843 (95% CI = 0.688 ~ 0.940, P < 0.001). However, the level of fecal F. n and fecal F. p remained unaltered between groups. Besides, mNGS showed that the pharyngeal swabs of COVID-19 patients were dominated by opportunistic pathogens. CONCLUSIONS: Pharyngeal but not fecal F. n was significantly increased in COVID-19 patients, clinicians should pay careful attention to potential coinfection. Pharyngeal F. n may serve as a promising candidate indicator for COVID-19.


Subject(s)
COVID-19/microbiology , Feces/microbiology , Fusobacterium Infections/microbiology , Fusobacterium nucleatum/genetics , Pharynx/microbiology , Adult , Biomarkers/analysis , COVID-19/virology , Carrier State/microbiology , Coinfection/microbiology , Coinfection/virology , Dysbiosis , Female , Fusobacterium Infections/virology , High-Throughput Nucleotide Sequencing , Humans , Male , Metagenomics , Microbiota , Middle Aged , Pharynx/virology , Sex Factors
3.
BMC Microbiol ; 21(1): 56, 2021 02 19.
Article in English | MEDLINE | ID: covidwho-1090700

ABSTRACT

BACKGROUND: Gastrointestinal symptoms are common in COVID-19 patients and SARS-CoV-2 RNA has been detected in the patients' feces, which could lead to fecal-oral transmission. Therefore, fecal sample testing with real-time RT-PCR is highly recommended as a routine test for SARS-CoV-2 infection. However, varying rates of detection in fecal sample have been reported. The aim of this study was to provide insights into the detection rates of SARS-CoV-2 in COVID-19 patients' fecal sample by using four real-time RT-PCR kits and two pretreatment methods (inactive and non-inactive). RESULTS: The detection rate of Trizol pretreatment group was slightly higher than that of Phosphate Buffered Saline (PBS) groups, showing that pretreatment and inactivation by Trizol had no influence to SARS-CoV-2 nucleic acid test (NAT) results. 39.29% detection rate in fecal sample by DAAN was obtained, while Bio-germ was 40.48%, Sansure 34.52%, and GeneoDx 33.33%. The former three kits had no significant difference. The DAAN kit detection rates of ORF1ab and N gene were nearly equal and Ct value distribution was more scattered, while the Bio-germ kit distribution was more clustered. The positive rate of SARS-COV-2 in fecal samples correlated with the severity of the disease, specifically, severe cases were less likely to be identified than asymptomatic infection in the DAAN group (adjusted OR 0.05, 95%CI = 0.00 ~ 0.91). CONCLUSIONS: Trizol should be of choice as a valid and safe method for pretreatment of fecal samples of SARS-CoV-2. All real-time RT-PCR kits assessed in this study can be used for routine detection of SARS-CoV-2 in fecal samples. While DAAN, with high NAT positive rate, could be the best out of the 4 kits used in this study. SARS-CoV-2 positive rate in fecal sample was related to the severity of illness.


Subject(s)
COVID-19/diagnosis , COVID-19/virology , Feces/microbiology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity , Adult , Female , Humans , Male , Middle Aged , Open Reading Frames/genetics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification
4.
Med Hypotheses ; 143: 109842, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-276450

ABSTRACT

The incidence of COVID-19 in children and teenagers is only about 2% in China. Children had mild symptoms and hardly infected other children or adults. It is worth considering that children are the most vulnerable to respiratory pathogens, but fatal SARS-like virus had not caused severe cases among them. According to the pathological studies of COVID-19 and SARS, a sharp decrease in T lymphocytes leads to the breakdown of the immune system. The cellular immune system of children differs from that of adults may be the keystone of atypical clinical manifestations or even covert infection. The frequent childhood vaccinations and repeated pathogens infections might be resulting in trained immunity of innate immune cells, immune fitness of adaptive immune cells or cross-protection of antibodies in the children. Therefore, due to lack of specific vaccine, some vaccines for tuberculosis, influenza and pneumonia may have certain application potential for the front-line health workers in the prevention and control of COVID-19. However, for high-risk susceptible populations, such as the elderly with basic diseases such as hypertension and diabetes, it is necessary to explore the remedial effect of the planned immune process on their immunity to achieve the trained immunity or immune fitness, so as to improve their own antiviral ability.


Subject(s)
Coronavirus Infections/epidemiology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/prevention & control , Vaccination , Adolescent , Antibodies, Viral/immunology , Betacoronavirus , COVID-19 , Child , Child, Preschool , China/epidemiology , Coinfection , Humans , Immunization Programs , Infant , SARS-CoV-2 , T-Lymphocytes/cytology
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