Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France.

Background The COVID-19 pandemic has altered organ and tissue donations as well as transplantation practices. SARS-CoV-2 serological tests could help in the selection of donors. We assessed COVID-19 seroprevalence in a population of tissue donors, at the onset of the outbreak in France, before systematic screening of donors for SARS-CoV-2 RNA. Methods 235 tissue donors at the Lille Tissue Bank between November 1, 2019 and March 16, 2020 were included. Archived serum samples were tested for SARS-CoV-2 antibodies using two FDA-approved kits. Results Most donors were at higher risks for severe COVID-19 illness including age over 65 years (142/235) and/or presence of co-morbidities (141/235). According to the COVID-19 risk assessment of transmission, 183 out of 235 tissue donors presented with a low risk level and 52 donors with an intermediate risk level of donor derived infection. Four out of the 235 (1.7%) tested specimens were positive for anti-SARS-CoV-2 antibodies: 2 donors with anti-N protein IgG and 2 other donors with anti-S protein total Ig. None of them had both type of antibodies. Conclusion Regarding the seroprevalence among tissue donors, we concluded that the transmission probability to recipient via tissue products was very low at the beginning of the outbreak.

Performance and economic evaluation of the molecular detection of pathogens for patients with severe infections: the EVAMICA open-label, cluster-randomised, interventional crossover trial.

PURPOSE: Microbiological diagnosis (MD) of infections remains insufficient. The resulting empirical antimicrobial therapy leads to multidrug resistance and inappropriate treatments. We therefore evaluated the cost-effectiveness of direct molecular detection of pathogens in blood for patients with severe sepsis (SES), febrile neutropenia (FN) and suspected infective endocarditis (SIE). METHODS: Patients were enrolled in a multicentre, open-label, cluster-randomised crossover trial conducted during two consecutive periods, randomly assigned as control period (CP; standard diagnostic workup) or intervention period (IP; additional testing with LightCycler®SeptiFast). Multilevel models used to account for clustering were stratified by clinical setting (SES, FN, SIE). RESULTS: A total of 1416 patients (907 SES, 440 FN, 69 SIE) were evaluated for the primary endpoint (rate of blood MD). For SES patients, the MD rate was higher during IP than during CP [42.6% (198/465) vs. 28.1% (125/442), odds ratio (OR) 1.89, 95% confidence interval (CI) 1.43-2.50; P < 0.001], with an absolute increase of 14.5% (95% CI 8.4-20.7). A trend towards an association was observed for SIE [35.4% (17/48) vs. 9.5% (2/21); OR 6.22 (0.98-39.6)], but not for FN [32.1% (70/218) vs. 30.2% (67/222), P = 0.66]. Overall, turn-around time was shorter during IP than during CP (22.9 vs. 49.5 h, P < 0.001) and hospital costs were similar (median, mean ± SD: IP €14,826, €18,118 ± 17,775; CP €17,828, €18,653 ± 15,966). Bootstrap analysis of the incremental cost-effectiveness ratio showed weak dominance of intervention in SES patients. CONCLUSION: Addition of molecular detection to standard care improves MD and thus efficiency of healthcare resource usage in patients with SES. ClinicalTrials.gov registration number: NCT00709358.

DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in urine.

We evaluated the DNA stability of Chlamydia trachomatis and Neisseria gonorrhoeae in 55 urine samples. Crossing threshold (Ct) values were highly similar after 3 to 14 days at room temperature (+0.002, P = 0.99). Consequently, it does not seem necessary to transfer urine specimens into a transport medium in less than 24 hours as recommended by manufacturers.

Superantigenic Yersinia pseudotuberculosis induces the expression of granzymes and perforin by CD4+ T cells.

Bacterial superantigens (SAgs) are immunostimulatory toxins that induce acute diseases mainly through the massive release of inflammatory cytokines. Yersinia pseudotuberculosis is the only Gram-negative bacterium known to produce a SAg (Y. pseudotuberculosis-derived mitogen [YPM]). This SAg binds major histocompatibility complex class II molecules on antigen-presenting cells and T cell receptors (TcR) bearing the variable region Vß3, Vß9, Vß13.1, or Vß13.2 (in humans) and Vß7 or Vß8 (in mice). We have previously shown that YPM exacerbates the virulence of Y. pseudotuberculosis in mice. With a view to understanding the mechanism of YPM's toxicity, we compared the immune response in BALB/c mice infected with a YPM-producing Y. pseudotuberculosis or the corresponding isogenic, SAg-deficient mutant. Five days after infection, we observed strong CD4(+) Vß7(+) T cell expansion and marked interleukin-4 (IL-4) production in mice inoculated with SAg-producing Y. pseudotuberculosis. These phenomena were correlated with the activation of ypm gene transcription in liver and spleen. A transcriptomic analysis revealed that the presence of YPM also increased expression of granzyme and perforin genes in the host's liver and spleen. This expression was attributed to a CD4(+) T cell subset, rather than to natural killer T (NKT) cells that display a TcR with a Vß region that is potentially recognized by YPM. Increased production of cytotoxic molecules was correlated with hepatotoxicity, as demonstrated by an increase in plasma alanine aminotransferase activity. Our results demonstrate that YPM activates a potentially hepatotoxic CD4(+) T cell population.

Virus and cystic fibrosis: rhinoviruses are associated with exacerbations in adult patients.

BACKGROUND: Few studies have suggested the potential role of respiratory viruses in cystic fibrosis (CF) exacerbation, but their real impact is probably underestimated. METHOD: Sixty-four sputum samples collected from 46 adult patients were included in the study: 33 samples were collected during exacerbation of CF, and 31 during the stable phase. After extraction, nucleic acids were tested for the presence of respiratory viruses. When rhinovirus (HRV) was detected, the 5'UTR and VP4/2 regions were sequenced, and phylogenetically analyzed. The characteristics of patients in exacerbation and stable phase were compared. RESULTS: Viruses were found in 25% of samples. The HRV viruses were the most frequently detected followed by coronaviruses. Only the HRV detection was significantly associated with the occurrence of CF pulmonary exacerbation (p<0.027). Characterization of 5'UTR and VP4/2 regions of the HRV genome specified that HRV-A, -B, -C were detected. All HRV-C were recombinant HRV-Ca. CONCLUSIONS: HRV were the most frequently detected viruses; their detection was significantly associated with the occurrence of an exacerbation. The reality of viral recombination between HRV was demonstrated in CF patients for the first time, raising the role of viruses in lung microbiota. Further studies are now warranted to decipher virus impact in CF.

Molecular methods in the diagnosis of Clostridium difficile infections: an update.

Nucleic acid amplification techniques (NAATs) represent a major advance in the diagnosis of Clostridium difficile (C. difficile) infection. This review analyzes the different options available for a molecular diagnosis of C. difficile infection, as well as the strengths and weaknesses of NAATs. The performances of seven commercials NAATs are compared (BD GeneOhm Cdiff, Illumigene C. difficile, Xpert C. difficile, BD Max Cdiff, Portrait Toxigenic C. difficile, ProGastro Cd, Seeplex Diarrhea ACE). The sensitivity and the rapidity of NAATs are excellent: additional efforts should focus on the discrimination between infection and colonization. Reporting the DNA load of toxigenic C. difficile in the stool sample may represent a solution. Diagnostic algorithms combining immunoassays and NAATs could also improve the specificity and reduce the global cost of this analysis.

PCR-electrospray ionization time-of-flight mass spectrometry: a new tool for the diagnosis of infective endocarditis from heart valves.

A polymerase chain reaction with an injection of the amplicons in an electrospray ionization mass spectrometry (PCR-ESI-MS) technique was evaluated for the diagnosis of bacterial and yeast pathogens on 13 cardiac valves with suspected endocarditis. At the moment of surgery, 3/13 PCR-ESI-MS results matched with microbiological documentation. Nine PCR-ESI-MS results correlated with Duke's criteria, leukocytes, C-reactive protein and blood cultures before surgery. The PCR-ESI-MS result of the last valve failed to confirm the blood culture result obtained fifteen days before. With speed and accuracy, this method may be useful to assert microbiological identification and adapt treatment.

Evaluation of a new molecular test, the BD Max Cdiff, for detection of toxigenic Clostridium difficile in fecal samples.

A new molecular assay detecting toxigenic Clostridium difficile, the BD Max Cdiff (Becton, Dickinson), was evaluated with 360 diarrheal feces samples. It exhibited high sensitivity (97.7%) and specificity (99.7%). The positive (97.7%) and negative (99.7%) predictive values of this test allow an accurate answer within 2 h.

Usefulness of real-time PCR for the diagnosis of sepsis in ICU-acquired infections.

Real-time PCR methods are able to rapidly detect a wide panel of microorganisms. These methods are of interest in critically ill patients to determine the presence of bacteria in the blood and other biological samples, especially in those patients with prior antimicrobial treatment. In intensive care unit (ICU), the LightCycler SeptiFast (LC-SF) Test provides 1.5 to 2 fold higher positivity rate compared with conventional blood cultures. Although identification of the bacterium by LC-SF is rapid and sensitive, susceptibility test could not be performed using this technique, except the methicillin- resistance for Staphylococci. The conventional cultures remain necessary for samples in ICU because of the high incidence of multidrug-resistant bacteria and the need for antimicrobial susceptibility of the bacterium to treat the patient correctly. A negative result for a Gram positive or negative bacterium allows deescalating the initial antimicrobial treatment, and decreasing the pressure of selection. Moreover, it is necessary to understand and interpret a DNA signal knowing that a dead bacterial material may be detected in a patient without any infection. What is the clinical relevance of bacterial DNA present in the blood and does the DNAemia found reflect true infection? Cost-effectiveness of the real-time PCR should be determined. Meanwhile, this test should be restricted to severe clinical situations, especially ICU patients with severe sepsis. In the future, real-time PCR tests should include more pathogens and antimicrobial resistant targets.

Application of quantitative PCR to the diagnosis and monitoring of Pseudomonas aeruginosa colonization in 5-18-year-old cystic fibrosis patients.

Early detection of Pseudomonas aeruginosa and early aggressive treatment are recommended to delay chronic infection in cystic fibrosis (CF) patients. The aim of this study was to assess a quantitative PCR (q-PCR) assay for the diagnosis of early P. aeruginosa colonization in 23 young CF patients (group A, age range 7-18 years) and to survey the eradication of P. aeruginosa in 10 young CF patients (group B, age range 5-18 years) after an initial antibiotic treatment. q-PCR results for consecutive sputum samples from each patient during a period of 18 months were compared with bacterial cultures during the same period plus an additional period of 12 months, and with concomitant clinical signs of pulmonary exacerbation. The q-PCR and bacterial cultures were negative for 17 of the 23 patients in group A and six of the 10 patients in group B during the study period. However, consecutive positive q-PCR results were observed for one patient in group A and three patients in group B, while the bacterial cultures for the same sputum sample remained negative. They preceded positive P. aeruginosa bacterial cultures at 7 and 8 months for two patients in group B. These positive results were associated with a worsening of the clinical status of patients, but pulmonary exacerbation appeared non-specific for the diagnosis of early P. aeruginosa colonization since pulmonary exacerbations were observed in patients in whom q-PCR or bacterial culture remained negative. In conclusion, q-PCR may be a useful additional tool to provide information on the P. aeruginosa status of CF patients.

Molecular diagnosis of a bilateral panuveitis due to Borrelia burgdorferi sensu lato by cerebral spinal fluid analysis.

The present paper describes a case of bilateral panuveitis due to Borrelia burgdorferi sensu lato diagnosed by a PCR approach using cerebral spinal fluid. Since the culture of B. burgdorferi takes a long time to grow and the accuracy of serological tests is doubtful in patients, the PCR method of amplifying a B. burgdorferi flagellin could be suitable to make a positive diagnosis in a case of atypical clinical history of Lyme disease.

Development and use of an internal positive control for detection of Bordetella pertussis by PCR.

An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS481.

Detection of Yersinia pestis in sputum by real-time PCR.

A 5' nuclease PCR assay for detection of the Yersinia pestis plasminogen activator (pla) gene in human respiratory specimens with simulated Y. pestis infection was developed. An internal positive control was added to the reaction mixture in order to detect the presence of PCR inhibitors that are often found in biological samples. The assay was 100% specific for Y. pestis. In the absence of inhibitors, a sensitivity of 10(2) CFU/ml of respiratory fluid was obtained. When inhibitors were present, detection of Y. pestis DNA required a longer sample treatment time and an initial concentration of bacteria of at least 10(4) CFU/ml. The test's total turnaround time was less than 5 h. The assay described here is well suited to the rapid diagnosis of pneumonic plague, the form of plague most likely to result from a bioterrorist attack.

The superantigen gene ypm is located in an unstable chromosomal locus of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3')-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.