Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19.

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.

Metatranscriptomic Characterization of Coronavirus Disease 2019 Identified a Host Transcriptional Classifier Associated With Immune Signaling.

BACKGROUND: The recent identification of a novel coronavirus, also known as severe acute respiratory syndrome coronavirus 2, has caused a global outbreak of respiratory illnesses. The rapidly developing pandemic has posed great challenges to diagnosis of this novel infection. However, little is known about the metatranscriptomic characteristics of patients with coronavirus disease 2019 (COVID-19). METHODS: We analyzed metatranscriptomics in 187 patients (62 cases with COVID-19 and 125 with non-COVID-19 pneumonia). Transcriptional aspects of 3 core elements, pathogens, the microbiome, and host responses, were evaluated. Based on the host transcriptional signature, we built a host gene classifier and examined its potential for diagnosing COVID-19 and indicating disease severity. RESULTS: The airway microbiome in COVID-19 patients had reduced alpha diversity, with 18 taxa of differential abundance. Potentially pathogenic microbes were also detected in 47% of the COVID-19 cases, 58% of which were respiratory viruses. Host gene analysis revealed a transcriptional signature of 36 differentially expressed genes significantly associated with immune pathways, such as cytokine signaling. The host gene classifier built on such a signature exhibited the potential for diagnosing COVID-19 (area under the curve of 0.75-0.89) and indicating disease severity. CONCLUSIONS: Compared with those with non-COVID-19 pneumonias, COVID-19 patients appeared to have a more disrupted airway microbiome with frequent potential concurrent infections and a special trigger host immune response in certain pathways, such as interferon-gamma signaling. The immune-associated host transcriptional signatures of COVID-19 hold promise as a tool for improving COVID-19 diagnosis and indicating disease severity.

[Metagenomic Sequencing for Pathogens Detection in a Critically Ill Patient].

OBJECTIVE: To detect pathogens in a critically ill patient using metagenomic sequencing. METHODS: A critically ill patient with severe acute pancreatitis suffered from abdominal pain and progressed into unconsciousness. Tissue smear, culture, automated biochemical identification and antibiotic susceptibility test, viral load determination by real-time fluorescence quantitative PCR, and immunohistochemical pathological tests were performed to detect pathogens, in addition to metagenomic sequencing based on the BGISEQ-100 high throughput sequencing platform. The sequences exclusive of host sequences were searched in the microbial genome database including viruses, bacteria, fungi and parasites. RESULTS: The patient was infected with methicillin-resistant Staphylococcus aureus, carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Acinetobacter baumannii, verified by both the routine methods and the metagenomic sequencing. The metagenomic sequencing also detected cytomegalovirus (CMV) with a turn-around time of 5 days. Real-time fluorescent quantitative PCR confirmed 189 000 copies/mL CMV load. CONCLUSION: In this case, three species of bacteria and one virus were detected by metagenomic sequencing quickly and accurately. Metagenomic sequencing may be helpful for diagnosing infectious diseases in critically ill patients.

Legionella pneumophila effector WipA, a bacterial PPP protein phosphatase with PTP activity.

The gram-negative bacterium Legionella pneumophila invades human's lung and causes Legionnaires' disease. To benefit its survival and replication in cellular milieu, L. pneumophila secrets at least 330 effector proteins into host cells. We found that the effector WipA has the protein tyrosine phosphatase (PTP) activity but does not depend on the classical CX5R motif for activity, suggesting that WipA is an unconventional PTP. Meanwhile, the presence of three other highly conserved motifs typically seen in protein serine/threonine phosphatases and the poor inhibition of WipA activity by okadaic acid led us to propose that WipA is a bacterial protein phosphatase. In addition, the determination of the 2.55-Å crystal structure of WipA revealed that WipA resembles cold-active protein tyrosine phosphatase (CAPTPase), and therefore very likely shares the same catalytic mechanism.

Trichomonas vaginalis in bronchoalveolar lavage fluid of a patient with severe pneumonia detected by metagenomic next-generation sequencing: A case report.

RATIONALE: Trichomonas vaginalis (T. vaginalis) is a common anaerobic parasitic protozoan. However, to the best of our knowledge, there are few reports documenting T. vaginalis infection outside the genitourinary tract. Severe pneumonia caused by T. vaginalis infection has been rarely reported. PATIENT CONCERNS: The 80-year-old female patient had a 20-year history of type II diabetes; however, she was not on regular medication. She was hospitalized due to a coma which continued 2 hours caused by trauma after a car accident. After her admission, she was provided with continuous mechanical ventilation; during the ventilation, she was still in a coma, accompanied by repeated fever and presence of much yellow sticky phlegm. The head CT scan indicated temporal lobe hematoma and subarachnoid hemorrhage. The lung CT scan showed bilateral pulmonary inflammatory consolidation and mass lesions. DIAGNOSES: She was initially diagnosed with severe pneumonia and acute respiratory distress syndrome. Subsequently, fiberoptic bronchoscopy was conducted, and bronchoalveolar lavage fluid (BALF) was collected and sent for metagenomic next-generation sequencing (mNGS). The result indicated the presence of abundant sequences from the T. vaginalis genome. Thus, she was diagnosed with pulmonary T. vaginalis infection. INTERVENTION: Anti-infective ornidazole treatment has significantly improved her symptoms. OUTCOMES: After treatment, the patient regained consciousness and was able to communicate, and there was no obvious expectoration, fever, or positive bronchus sign in the lungs. Thereby, she was discharged from the hospital. LESSONS: Special attention should be paid to infections other than common bacterial infections, such as T. vaginalis. Moreover, infection of rare pathogenic microorganisms might show symptoms similar to common bacterial infection, leading to misdiagnosis, further highlighting the usefulness of mNGS in detecting pathogens in a timely, sensitive, and accurate manner.