A semi-automated protocol for Archaea DNA extraction from stools

Background: The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens. Findings: We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qiagen DNA bacteria card and the EZ1 ® DNA Tissue Kit (Qiagen, Courtaboeuf, France) yielded 35/110 (32%) positives for the real-time PCR detection of the Methanobrevibacter smithii 16S rRNA gene, with average Ct values of 36.1. A semi-automated protocol combining glass-powder crushing, overnight proteinase K digestion and lysis in the buffer from the EZ1 kit yielded 90/110 (82%) positive specimens (P = 0.001) with an average Ct value of 27.4 (P = 0.001). The manual protocol yielded 100/110 (91%) positive specimens (P = 0.001) with an average Ct value of 30.33 (P = 0.001). However, neither the number of positive specimens nor the Ct values were significantly different between the manual protocol and the semiautomated protocol (P> 0.1 and P> 0.1). Conclusion: Proteinase K digestion and glass powder crushing dramatically increase the extraction yield of archaea DNA from human stools. The semi-automated protocol described here was more rapid than the manual protocol and yielded significantly more archaeal DNA. It could be applied for extracting total stool DNA for further PCR amplification

Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

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Isolation of Viable SARS-CoV-2 Virus from Feces of an Immunocompromised Patient Suggesting a Possible Fecal Mode of Transmission

International audience(1) Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) excretion in stools is well documented by RT-PCR, but evidences that stools contain infectious particles are scarce. (2) Methods: After observing a Corona Virus 2019 Disease (COVID-19) epidemic cluster associated with a ruptured sewage pipe, we search for such a viable SARS-CoV-2 particle in stool by inoculating 106 samples from 46 patients. (3) Results: We successfully obtained two isolates from a unique patient with kidney transplantation under immunosuppressive therapy who was admitted for severe diarrhea. (4) Conclusions: This report emphasizes that SARS-CoV-2 is an enteric virus, and infectious virus particles can be isolated from the stool of immune-compromised patients like, in our case, kidney transplant recipient. Immune-compromised patients are likely to have massive multiplication of the virus in the gastrointestinal tract and this report suggests possible fecal transmission of SARS-CoV-2

<it>Haemophilus pittmaniae</it> respiratory infection in a patient with siderosis: a case report

Abstract Introduction Haemophilus pittmaniae was described in 2005 as a new species distantly related to Haemophilus parainfluenzae. This member of the human saliva microbiota has also been further isolated from various body fluids without formal description of the patients. Case presentation We report the case of H. pittmaniae isolate made from a sputum specimen collected from a 58-year-old Caucasian man with a massive fibrotic form of siderosis who was awaiting lung transplantation. Identification of the isolate was ascertained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene sequencing. H. pittmaniae was considered to be responsible for the worsening of the patient’s chronic respiratory failure and was successfully treated with oral amoxicillin. Conclusion H. pittmaniae should be regarded as a new pathogen responsible for respiratory tract infection in patients with chronic lung diseases.</p

Haemophilus pittmaniae respiratory infection in a patient with siderosis: a case report

International audienceHaemophilus pittmaniae was described in 2005 as a new species distantly related to Haemophilus parainfluenzae. This member of the human saliva microbiota has also been further isolated from various body fluids without formal description of the patients. Case presentation: We report the case of H. pittmaniae isolate made from a sputum specimen collected from a 58-year-old Caucasian man with a massive fibrotic form of siderosis who was awaiting lung transplantation. Identification of the isolate was ascertained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene sequencing. H. pittmaniae was considered to be responsible for the worsening of the patient's chronic respiratory failure and was successfully treated with oral amoxicillin

Culturomics Discloses Anti-Tubercular Enterococci Exclusive of Pulmonary Tuberculosis: A Preliminary Report

Mycobacterium tuberculosis causes pulmonary tuberculosis, a deadly infection of which the clinical expression and prognosis are not fully understood at the individual level, apart from genetic susceptibility traits. We investigated whether individual gut microbiota may correlate with pulmonary tuberculosis status. Culturomics investigations of gut microbiota in two pulmonary tuberculosis patients and two controls in Burkina Faso found 60 different bacterial species in patients and 97 in controls, including 45 in common. Further analysis of the results at the individual level indicated seven bacteria, including Enterococcus mundtii and Enterococcus casseliflavus, which were exclusively cultured in controls. Blind quantitative PCR-based exploration of faeces samples in two cohorts in Burkina Faso and in France confirmed a nonsignificant association of E. mundtii and E. casseliflavus with controls. Further in vitro explorations found four E. mundtii and E. casseliflavus strains inhibiting the growth of M. tuberculosis strains representative of four different lineages as well as Mycobacterium africanum, Mycobacterium canettii, and Mycobacterium bovis, in an inoculum-dependent manner. Heat-killed E. mundtii or E. casseliflavus were ineffective. These unprecedented observations of direct interactions between gut E. mundtii and E. casseliflavus with M. tuberculosis complex mycobacteria suggest that gut microbiota may modulate the expression of pulmonary tuberculosis

Prevalence of Mycobacterium lentiflavum in cystic fibrosis patients, France

International audienceBackground: Mycobacterium lentiflavum is rarely isolated in respiratory tract samples from cystic fibrosis patients. We herein describe an unusually high prevalence of M. lentiflavum in such patients. Methods: M. lentiflavum, isolated from the respiratory tract of cystic fibrosis patients, was identified using both rpoB partial sequencing and detected directly in the sputum by using real-time PCR targeting the smpB gene. Results: M. lentiflavum emerged as the third most prevalent nontuberculous mycobacterial species isolated in cystic fibrosis patients in Marseille, France. Six such patients were all male, and two of them may have fulfilled the American Thoracic Society clinical and microbiological criteria for M. lentiflavum potential lung infection. Conclusions: M. lentiflavum was the third most common mycobacteria isolated in cystic fibrosis patients, particularly in six male patients. M. lentiflavum outbreaks are emerging particularly in cystic fibrosis patients

Sequential Appearance and Isolation of a SARS-CoV-2 Recombinant between Two Major SARS-CoV-2 Variants in a Chronically Infected Immunocompromised Patient

International audienceGenetic recombination is a major evolutionary mechanism among RNA viruses, and it is common in coronaviruses, including those infecting humans. A few SARS-CoV-2 recombinants have been reported to date whose genome harbored combinations of mutations from different mutants or variants, but only a single patient’s sample was analyzed, and the virus was not isolated. Here, we report the gradual emergence of a hybrid genome of B.1.160 and Alpha variants in a lymphoma patient chronically infected for 14 months, and we isolated the recombinant virus. The hybrid genome was obtained by next-generation sequencing, and the recombination sites were confirmed by PCR. This consisted of a parental B.1.160 backbone interspersed with two fragments, including the spike gene, from an Alpha variant. An analysis of seven sequential samples from the patient decoded the recombination steps, including the initial infection with a B.1.160 variant, then a concurrent infection with this variant and an Alpha variant, the generation of hybrid genomes, and eventually the emergence of a predominant recombinant virus isolated at the end of the patient’s follow-up. This case exemplifies the recombination process of SARS-CoV-2 in real life, and it calls for intensifying the genomic surveillance in patients coinfected with different SARS-CoV-2 variants, and more generally with several RNA viruses, as this may lead to the appearance of new viruses

Methanobrevibacter smithii Archaemia in Febrile Patients With Bacteremia, Including Those With Endocarditis

International audienceAbstract Background The spectrum of infections caused by methanogens remains to be described. We searched for methanogens in the blood of febrile patients using specific tools. Methods Blood culture samples routinely collected in patients with fever were prospectively screened by specific PCR assays for methanogens. Positive samples were observed by autofluorescence and electron microscopy, analyzed by metagenomics and cultured using previously developed methods. Blood culture bottles experimentally inoculated were used as controls. The presence of methanogens in vascular and cardiac tissues was assessed by indirect immunofluorescence, fluorescent in situ hybridization and PCR-based investigations. Results PCR detection attempted in 7,716 blood samples, was negative in all 1,312 aerobic bottles and 810 bacterial culture-negative anaerobic bottles. PCRs were positive in 27/5,594 (0.5%) bacterial culture-positive anaerobic bottles collected from 26 patients. Sequencing confirmed Methanobrevibacter smithii associated with staphylococci in 14 patients, Enterobacteriaceae in nine patients and streptococci in three patients. Metagenomics confirmed M. smithii in five samples, and M. smithii was isolated in broth from two samples; the genomes of these two isolates were sequenced. Blood cultures experimentally inoculated with Enterobacteriaceae, Staphylococcus epidermidis or Staphylococcus hominis yielded hydrogen, but no methane, authentifying observational data. Three patients diagnosed with infectious mitral endocarditis, were indisputably diagnosed by microscopy, PCR-based detections and culture: we showed M. smithii microscopically and by a specific PCR followed by sequencing method in two of three cardiovascular tissues. Conclusions Using appropriate laboratory methods, M. smithii is demonstrated as causing archaemia and endocarditis in febrile patients who are coinfected by bacteria