Evaluation of the Performance Characteristics of a New POC Multiplex PCR Assay for the Diagnosis of Viral and Bacterial Neuromeningeal Infections.

Point-of-care syndromic PCR (POC SPCR) assays are useful tools for the rapid detection of the most common causative agents of community-acquired infections responsible for meningitis and encephalitis infections. We evaluated the performance characteristics of the new QIAstat-Dx® Meningitis/Encephalitis panel (QS) compared to the laboratory reference methods and the POC SPCR Biofire® FilmArray® Meningitis Encephalitis Panel (FA). Viral (Enterovirus, Parechovirus, HSV-1, HSV-2, HHV-6, VZV) and bacterial (E. coli K1, H. influenzae, L. monocytogenes, encapsulated N. meningitidis, M. pneumoniae, S. agalactiae, S. pneumoniae, S. pyogenes) pathogens were suspended at low concentrations and tested with the POC SPCR systems. The reproducibility, analytical specificity, carryover contamination, interferences and clinical samples were evaluated. All samples tested positive with both QS and FA except for those containing the lowest concentrations of Enterovirus-D68-B3, Echovirus-30 and S. agalactiae which were only detected by FA. In terms of analytical specificity, we observed 3 false positive results out of 48 QS tests versus 1 out of 37 FA tests. For the other studied criteria, both QS and FA performed as expected. Our results suggest that the performance characteristics of QS are close to those of FA. A prospective multicenter study would be useful to complete the performances evaluation of QS.

Both microbiological surveillance and audit of procedures improve reprocessing of flexible bronchoscopes and patient safety.

BACKGROUND: Microbiological surveillance of bronchoscopes and automatic endoscope reprocessors (AERs)/washer disinfectors as a quality control measure is controversial. Experts also are divided on the infection risks associated with bronchoscopic procedures. OBJECTIVE: We evaluated the impact of routine microbiological surveillance and audits of cleaning/disinfection practices on contamination rates of reprocessed bronchoscopes. DESIGN: Audits were conducted of reprocessing procedures and microbiological surveillance on all flexible bronchoscopes used from January 2007 to June 2020 at a teaching hospital in France. Contamination rates per year were calculated and analyzed using a Poisson regression model. The risk factors for microbiological contamination were analyzed using a multivariable logistical regression model. RESULTS: In total, 478 microbiological tests were conducted on 91 different bronchoscopes and 57 on AERs. The rate of bronchoscope contamination significantly decreased between 2007 and 2020, varying from 30.2 to 0% (P < .0001). Multivariate analysis confirmed that retesting after a previous contaminated test was significantly associated with higher risk of bronchoscope contamination (OR, 2.58; P = .015). This finding was explained by the persistence of microorganisms in bronchoscopes despite repeated disinfections. However, the risk of persistent contamination was not associated with the age of the bronchoscope. CONCLUSIONS: Our results confirm that bronchoscopes can remain contaminated despite repeated reprocessing. Routine microbial testing of bronchoscopes for quality assurance and audit of decontamination and disinfection procedures can improve the reprocessing of bronchoscopes and minimize the rate of persistent contamination.

Priming with intranasal lactobacilli prevents Pseudomonas aeruginosa acute pneumonia in mice.

BACKGROUND: Increasing resistance to antibiotics of Pseudomonas aeruginosa leads to therapeutic deadlock and alternative therapies are needed. We aimed to evaluate the effects of Lactobacillus clinical isolates in vivo, through intranasal administration on a murine model of Pseudomonas aeruginosa pneumonia. RESULTS: We screened in vitro 50 pulmonary clinical isolates of Lactobacillus for their ability to decrease the synthesis of two QS dependent-virulence factors (elastase and pyocyanin) produced by Pseudomonas aeruginosa strain PAO1. Two blends of three Lactobacillus isolates were then tested in vivo: one with highly effective anti-PAO1 virulence factors properties (blend named L.rff for L. rhamnosus, two L. fermentum strains), and the second with no properties (blend named L.psb, for L. paracasei, L. salivarius and L. brevis). Each blend was administered intranasally to mice 18 h prior to PAO1 pulmonary infection. Animal survival, bacterial loads, cytological analysis, and cytokines secretion in the lungs were evaluated at 6 or 24 h post infection with PAO1. Intranasal priming with both lactobacilli blends significantly improved 7-day mice survival from 12% for the control PAO1 group to 71 and 100% for the two groups receiving L.rff and L.psb respectively. No mortality was observed for both control groups receiving either L.rff or L.psb. Additionally, the PAO1 lung clearance was significantly enhanced at 24 h. A 2-log and 4-log reduction was observed in the L.rff + PAO1 and L.psb + PAO1 groups respectively, compared to the control PAO1 group. Significant reductions in neutrophil recruitment and proinflammatory cytokine and chemokine secretion were observed after lactobacilli administration compared to saline solution, whereas IL-10 production was increased. CONCLUSIONS: These results demonstrate that intranasal priming with lactobacilli acts as a prophylaxis, and avoids fatal complications caused by Pseudomonas aeruginosa pneumonia in mice. These results were independent of in vitro anti-Pseudomonas aeruginosa activity on QS-dependent virulence factors. Further experiments are required to identify the immune mechanism before initiating clinical trials.

Rapid multiplex PCR assays in patients with respiratory viral infections: is semi-quantitative data useful? A pilot study.

Viral respiratory rapid multiplex PCR assays FilmArray® (FA) and ePlex® (eP) provide qualitative results which may not reflect clinical relevance. In a pilot study, we report retrospectively whether the semi-quantitative PCR assay R-GENE® would have facilitated clinical interpretation. Forty-four patients were hospitalized for various respiratory manifestations; all of them have benefited from a respiratory sample during acute symptoms. Among the 44 patients, FA detected 23 positive samples including 31 viruses, 26 of them gave high or moderate R-GENE® scores (cycle threshold < 35), and all but one were consistent with clinical history. Semi-quantitative scores would have allowed for critical interpretation of the results; those are a key additional element for an optimal exploitation of the rapid multiplex PCR assays power.

Prolonged 59-day course of COVID-19: The case of a SARS-CoV-2 shedding persistency in a healthcare provider.

For health professionals, repeated SARS-CoV-2 RNA detection through PCR combined with properly validated serologic assays for the presence of anti-IgM and anti-IgG antibodies should be mandatory.

Sputum versus nasopharyngeal samples for the molecular diagnosis of respiratory viral infection in cystic fibrosis: A pilot study.

Viruses are important agents in lung function deterioration in Cystic Fibrosis (CF). To date, no standard operating procedures (SOPs) have been established to determine which sampling method is the most effective for an optimal virological diagnosis of respiratory viral infections in CF. Here we investigated the performances of two sampling sites, sputum samples versus nasopharyngeal (NP) swabs, for thirty participants from three CF centres presenting an acute respiratory infection. Sputum and NP samples were simultaneously collected and multiplex PCR targeting 16 to 18 viruses were performed. Viruses were detected for 18/30 patients (60%). A high concordance between the sputum and NP samples was observed in 25 (83%) paired samples of which 13 tested positive and 12 tested negative. These results highlighted the relevance of sputum sampling for diagnostic of respiratory viruses in CF, which is less invasive and better accepted by CF patients than NP, and allows accurate bacterial detection.

Evaluation of posaconazole antifungal prophylaxis in reducing the incidence of invasive aspergillosis in patients with acute myeloid leukemia.

PURPOSE OF THE STUDY: Invasive aspergillosis (IA) is the most prevalent invasive fungal disease (IFD) in neutropenic patients. Environment is the main source of Aspergillus spores aerosolization especially during building construction. International guidelines recommend mechanical protection during hospital building works; otherwise the use of antifungal prophylaxis is not clearly indicated. Our objective was to determine the efficacy of antifungal prophylaxis by posaconazole on IA incidence in acute myeloid leukemia population and to analyse the benefit of this prophylaxis and HEPA-filters during hospital buildings works. PATIENTS AND METHODS: We included patients treated for acute myeloid leukemia at Brest teaching hospital from January 2009 to December 2015. We compared incidence of IA in the group treated by posaconazole from 2012 to 2015 to the incidence of IA in the first group who did not receive antifungal prophylaxis (from 2009 to 2011). The one-year overall survival was also analyzed using the Kaplan-Meier method. RESULTS: 245 patients were enrolled including 151 treated with posaconazole. 23 IA were diagnosed between 2009 and 2011 (without antifungal prophylaxis), then 31 between 2012 and 2015 (with posaconazole) without statistical difference between the incidence densities (0.34 per 100 hospitalization-days vs. 0.30 per 100 hospitalization-days, p = 0.71). Incidence density of IA increased during building works (2.40 per 100 hospitalization-days vs. 0.28 per 100 hospitalization-days, p < 0.0001). The incidence density of IA significantly decreased during construction periods when posaconazole prophylaxis was used (1.59 per 100 hospitalization-days vs. 4.87 per 100 hospitalization-days p < 0.0001). CONCLUSION: Our study suggests, for the first time, the interest of antifungal prophylaxis in addition to HEPA filtration in prevention of IA during hospital building works.

Natural non-homologous recombination led to the emergence of a duplicated V3-NS5A region in HCV-1b strains associated with hepatocellular carcinoma.

BACKGROUND: The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies. METHODS: Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplication-like event. RESULTS: Quasispecies were entirely composed of exclusively mutant or wild-type strains respectively. Mutant quasispecies were found to have been present since contamination and had persisted for at least 10 years. This V3 duplication-like event appears to have resulted from non-homologous recombination between HCV-1b wild-type strains around 100 years ago. The association between increased liver disease severity and these HCV-1b mutants may explain their persistence in chronically infected patients. CONCLUSIONS: These results emphasize the possible consequences of non-homologous recombination in the emergence and severity of new viral diseases.

Viruses in cystic fibrosis patients' airways.

Although bacteria have historically been considered to play a major role in cystic fibrosis (CF) airway damage, a strong impact of respiratory viral infections (RVI) is also now recognized. Emerging evidence confirms that respiratory viruses are associated with deterioration of pulmonary function and exacerbation and facilitation of bacterial colonization in CF patients. The aim of this review is to provide an overview of the current knowledge on respiratory viruses in CF airways, to discuss the resulting inflammation and RVI response, to determine how to detect the viruses, and to assess their clinical consequences, prevalence, and interactions with bacteria. The most predominant are Rhinoviruses (RVs), significantly associated with CF exacerbation. Molecular techniques, and especially multiplex PCR, help to diagnose viral infections, and the coming rise of metagenomics will extend knowledge of viral populations in the complex ecosystem of CF airways. Prophylaxis and vaccination are currently available only for Respiratory syncytial and Influenza virus (IV), but antiviral molecules are being tested to improve CF patients' care. All the points raised in this review highlight the importance of taking account of RVIs and their potential impact on the CF airway ecosystem.

Measures to improve microbial quality surveillance of gastrointestinal endoscopes.

BACKGROUND AND STUDY AIM: Infectious outbreaks associated with the use of gastrointestinal endoscopes have increased in line with the spread of highly resistant bacteria. The aim of this study was to determine the measures required to improve microbial quality surveillance of gastrointestinal endoscopes. METHODS: We reviewed the results of all microbiological surveillance testing of gastrointestinal endoscopes and automatic endoscope reprocessors (AERs) performed at Brest Teaching Hospital from 1 January 2008 to 1 June 2015. We analyzed the influence of the time of incubation on the rate of positive results using the Kaplan - Meier method. We also studied risk factors for gastrointestinal endoscope contamination using a multivariable logistic regression model. RESULTS: Over the study period, 1100 microbiological tests of gastrointestinal endoscopes (n = 762) and AERs (n = 338) were performed. A total of 264 endoscope tests (34.6 %) showed a level of contamination higher than the target. After 2 days of incubation, contamination was apparent in only 55.5 % of the endoscopes that were later shown to be contaminated (95 % confidence interval [CI] 49.2 - 61.8). Multivariable analysis showed that the use of storage cabinets for heat-sensitive endoscopes significantly reduced the risk of endoscope contamination (odds ratio [OR] 0.23, 95 %CI 0.09 - 0.54; P  < 0.001) and that the use of endoscopes older than 4 years significantly increased this risk (OR ≥ 6 vs. < 2 years 2.92, 95 %CI 1.63 - 5.24; P < 0.001). CONCLUSIONS: Microbiological culture technique, mainly incubation duration, strongly influenced the results of endoscope sampling. Samples should be cultured for more than 2 days to improve the detection of contaminated endoscopes. Particular attention should be paid to endoscopes older than 2 years and to those that are not stored in storage cabinets for heat-sensitive endoscopes.

Duplication of the V3 domain in hepatitis C virus (1b) NS5A protein: Clonal analysis and physicochemical properties related to hepatocellular carcinoma occurrence.

BACKGROUND: Hepatitis C virus non-structural protein 5A is known to play a role in development of hepatocellular carcinoma (HCC) via interactions with host cell pathways. OBJECTIVES: Hepatitis C virus genotype 1b strains presenting a wide insertion of 31 amino acids in the non-structural protein 5A V3 domain (V3 DI) were studied to determine whether this V3-like additional domain (V3 DII) was associated with HCC occurrence. STUDY DESIGN: Seventy-four patients' sera were screened for V3 DII presence regarding clinical status. RESULTS: Three strains with duplicated V3 were detected among patients with progression to HCC (n=28), two strains among patients with liver cirrhosis (Ci, n=27) and none among patients with chronic hepatitis (Chr, n=19). Phylogenetic trees built from V3 DI and V3 DII sequences indicated that the latter clustered separately. In between-group clonal analysis, V3 DII sequences from the HCC group were found to be more distant from HCV-J than V3 DI sequences (p<0.0001). Between-group comparisons showed significant differences in genetic distances from HCV-J, in HCC V3 DI and HCC V3 DII compared to Ci V3 DI and Ci V3 DII sequences (p<0.0001). HCC V3 DII domain and its junction with V3 DI exhibited higher Shannon entropy values and enrichment in disorder-promoting residues. CONCLUSIONS: Taken together, our results suggest that V3 DII evolution may differ in strains associated with HCC occurrence. The presence of an intrinsically "disordered" V3 duplicate may alter the NS5A protein network. Further investigations are necessary to elucidate the potential impact of V3 duplication in the context of carcinogenesis.

Classification algorithm for subspecies identification within the Mycobacterium abscessus species, based on matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Mycobacterium abscessus, as a species, has been increasingly implicated in respiratory infections, notably in cystic fibrosis patients. The species comprises 3 subspecies, which can be difficult to identify. Since they differ in antibiotic susceptibility and clinical relevance, developing a routine diagnostic tool discriminating Mycobacterium abscessus at the subspecies level is a real challenge. Forty-three Mycobacterium abscessus species isolates, previously identified by multilocus sequence typing, were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A subspecies identification algorithm, based on five discriminating peaks, was drawn up and validated by blind identification of a further 49 strains, 94% of which (n = 46) were correctly identified. Two M. abscessus subsp. massiliense strains were misidentified as M. abscessus subsp. abscessus, and for 1 other strain identification failed. Inter- and intralaboratory reproducibility tests were conclusive. This study presents, for the first time, a classification algorithm for MALDI-TOF MS identification of the 3 M. abscessus subspecies. MALDI-TOF MS proved effective in discriminating within the M. abscessus species and might be easily integrated into the workflow of microbiology labs.

Proposal of a quantitative PCR-based protocol for an optimal Pseudomonas aeruginosa detection in patients with cystic fibrosis.

BACKGROUND: The lung of patients with cystic fibrosis (CF) is particularly sensitive to Pseudomonas aeruginosa. This bacterium plays an important role in the poor outcome of CF patients. During the disease progress, first acquisition of P. aeruginosa is the key-step in the management of CF patients. Quantitative PCR (qPCR) offers an opportunity to detect earlier the first acquisition of P. aeruginosa by CF patients. Given the lack of a validated protocol, our goal was to find an optimal molecular protocol for detection of P. aeruginosa in CF patients. METHODS: We compared two formerly described qPCR formats in early detection of P. aeruginosa in CF sputum samples: a qPCR targeting oprL gene, and a multiplex PCR targeting gyrB and ecfX genes. RESULTS: Tested in vitro on a large panel of P. aeruginosa isolates and others gram-negative bacilli, oprL qPCR exhibited a better sensitivity (threshold of 10 CFU/mL versus 730 CFU/mL), whereas the gyrB/ecfX qPCR exhibited a better specificity (90% versus 73%). These results were validated ex vivo on 46 CF sputum samples positive for P. aeruginosa in culture. Ex vivo assays revealed that qPCR detected 100 times more bacterial cells than culture-based method did. CONCLUSION: Based on these results, we proposed a reference molecular protocol combining the two qPCRs, which offers a sensitivity of 100% with a threshold of 10 CFU/mL and a specificity of 100%. This combined qPCR-based protocol can be adapted and used for other future prospective studies.

Algorithm-based prediction of HIV-1 subtype D coreceptor use.

We compared the coreceptor tropism-predicting performance of a specific genotypic algorithm for HIV-1 subtype D and that of the geno2pheno algorithm with different cutoffs. The D-specific algorithm and geno2pheno with a false-positivity rate cutoff of 2.5% had the same concordance with the phenotypic determination. The geno2pheno algorithm with a false-positivity rate cutoff of 2.5%, more sensitive but slightly less specific, seems to be an appropriate alternative.

Multicenter quality control of hepatitis C virus protease inhibitor resistance genotyping.

Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

NS3 protease polymorphism and natural resistance to protease inhibitors in French patients infected with HCV genotypes 1-5.

BACKGROUND: Resistant HCV populations may pre-exist in patients before NS3 protease inhibitor therapy and would likely be selected under specific antiviral pressure. The higher prevalence and lower rate of response to treatment associated with HCV genotype 1 infections has led to drug discovery efforts being focused primarily on enzymes produced by this genotype. Protease inhibitors may also be useful for non-genotype-1-infected patients, notably for non-responders. METHODS: We investigated the prevalence of dominant resistance mutations and polymorphism in 298 HCV protease-inhibitor-naive patients infected with HCV genotypes 1, 2, 3, 4 or 5. Genotype-specific NS3 primers were designed to amplify and sequence the NS3 protease gene. RESULTS: None of the 233 analysed sequences contained major telaprevir (TVR) or boceprevir (BOC) resistance mutations (R155K/T/M, A156S/V/T and V170A). Some substitutions (V36L, T54S, Q80K/R, D168Q and V170T) linked to low or moderate decreases in HCV sensitivity to protease inhibitors were prevalent according to genotype (between 2% and 100%). Other than genotype signature mutations at positions 36, 80 and 168, the most frequent substitution was T54S (4 genotype 1 and 2 genotype 4 sequences). All genotype 2-5 sequences had the non-genotype-1 signature V36L mutation known to confer low-level resistance to both TVR and BOC. CONCLUSIONS: We have developed an HCV protease NS3 inhibitor resistance genotyping tool suitable for use with HCV genotypes 1-5. Polymorphism data is valuable for interpreting genotypic resistance profiles in cases of failure of anti-HCV NS3 protease treatment.