Complementarity between targeted real-time specific PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases.

Molecular tools have shown an added value in the diagnosis of infectious diseases, in particular for those caused by fastidious intracellular microorganisms, or in patients receiving antibiotics before sampling. If 16S rDNA amplification had been gradually implemented in microbiology laboratories, specific real-time polymerase chain reaction (PCR) would have permitted an increase in the sensitivity of molecular methods and a reduction of contamination. Herein, we report our experience in the diagnosis of infectious diseases over two years, during which 32,948 clinical samples from 18,056 patients were received from France and abroad. Among these samples, 81,476 PCRs were performed, of which 1,192 were positive. Molecular techniques detected intracellular microorganisms in 31.3 % of respiratory samples, 27.8 % of endocarditis samples and 51.9 % of adenitis samples. Excluding intracellular bacteria, 25 % of the positive samples in this series were sterile in culture. Conventional broad-range PCR permitted the identification of fastidious and anaerobic microorganisms, but specific real-time PCR showed a significant superiority in the diagnosis of osteoarticular infections, in particular for those caused by Kingella kingae and Staphylococcus aureus, and for endocarditis diagnosis, specifically when Streptococcus gallolyticus and Staphylococcus aureus were involved. The sensitivity of conventional broad-range PCR was 62.9 % concerning overall diagnoses for which both techniques had been performed. These findings should lead microbiologists to focus on targeted specific real-time PCR regarding the clinical syndrome. Finally, syndrome-driven diagnosis, which consists of testing a panel of microorganisms commonly involved for each syndrome, permitted the establishment of 31 incidental diagnoses.

Looking in ticks for human bacterial pathogens.

Ticks are considered to be second worldwide to mosquitoes as vectors of human diseases and the most important vectors of disease-causing pathogens in domestic and wild animals. A number of emerging tick-borne pathogens are already discovered; however, the proportion of undiagnosed infectious diseases, especially in tropical regions, may suggest that there are still more pathogens associated with ticks. Moreover, the identification of bacteria associated with ticks may provide new tool for the control of ticks and tick-borne diseases. Described here molecular methods of screening of ticks, extensive use of modern culturomics approach, newly developed artificial media and different cell line cultures may significantly improve our knowledge about the ticks as the agents of human and animal pathology.

Molecular diagnosis of bacterial vaginosis: impact on IVF outcome.

Bacterial vaginosis can increase obstetrical complications such as miscarriage, premature rupture of membranes and preterm delivery. The aim of our study was first to assess BV prevalence for infertile patients treated by in vitro fertilisation (IVF) using both the Nugent score and polymerase chain reaction (PCR), and then to assess the impact of BV on the pregnancy rate after IVF. Vaginal samples were obtained from women followed for IVF in our Assisted Reproduction Technology (ART) Unit between August 2010 and April 2011. For each patient, two techniques were performed to diagnose BV: Gram staining to assess the Nugent score and a quantitative molecular analysis using a specific real-time PCR assay. Two groups were studied: normal flora (BV-) and BV (BV+). The primary outcome measure was the implantation rate. The secondary outcomes were clinical pregnancy rate, early and late miscarriage, premature rupture of membranes, preterm delivery, mode of delivery and birthweight. A total of 307 patients were included. PCR revealed a prevalence of BV of 9.45 %. Among women who performed vaginal douching, 22.2 % were BV+, whereas 7.9 % of patients who did not douche were BV+ (p = 0.028). The embryo implantation rate was decreased between the BV- and BV+ groups (36.3 % vs. 27.6 %, p = 0.418), but it was not significant. Obstetrical outcomes did not present significant statistical differences among the groups. Vaginal douching significantly enhanced BV in women treated with IVF. We also observed a non-significant decrease of embryo implantation rate and clinical pregnancy rate for women treated by IVF.

Outbreak of adenovirus D8 in a neonatal intensive care unit involving multiple simultaneous transmission pathways.

BACKGROUND: Adenovirus (ADV) outbreaks in neonatal intensive care units (NICU) can lead to durable transmission and serious adverse outcomes. This study describes the investigation and control of an ADV-D8 outbreak in an NICU, associated with ophthalmologic equipment used during retinopathy of prematurity (ROP) screening. Cases were observed in neonates, parents and nurses. METHODS: The outbreak investigation was performed including sampling patients, parents and health care workers as well as the environment for molecular detection of ADV DNA. The investigation was also conducted in the guest house where some parents were temporary residents. A retrospective cohort study focused on neonates hospitalized during the epidemic period to assess the risk associated with ROP examination. RESULTS: Fifteen cases were identified in neonates; all but one presented with conjunctivitis. Two healthcare workers and 18 parents acquired conjunctivitis. ADV DNA was identified on the RetCam and on the freezer shared by parents. All ADV-positive samples were typed as ADV-D8. ADV infections occurred more frequently in neonates who had ROP examinations (37.8% (14/37) vs (0.9% (1/110); P<0.001) (relative risk 41.6; (5.7-305.8)). The RetCam was disinfected between two examinations using a disinfectant that was virucidal on ADV after a 30-min contact. CONCLUSION: This outbreak was significantly associated with ROP examination with a RetCam that had a disinfection protocol ill-adapted to rapid patient turnover. In addition, nosocomial transmission via the parents to neonates and parent-to-parent transmission is likely to have played a role in the dissemination of cases. No further cases were observed after the new disinfection procedure was enforced.

Self-collected vaginal swabs for the quantitative real-time polymerase chain reaction assay of Atopobium vaginae and Gardnerella vaginalis and the diagnosis of bacterial vaginosis.

The aim of this study was to assess the feasibility of using self-collected vaginal specimens for the quantitative real-time polymerase chain reaction (qPCR) assays of bacterial vaginosis (BV)-associated bacteria versus practitioner-collected swabs. A cross-sectional study included 190 pregnant women enrolled before 20 weeks' gestation from September 2008 to November 2009. Self- and practitioner-collected swabs were taken during the same prenatal visit for each woman, qPCR assays performed for each, and the results compared. The quantification of the human albumin gene was used as an internal control to ensure sampling quality and accurate comparisons. The level of agreement of the qPCR assays for each microorganism was calculated with the Spearman product moment correlation coefficient and the kappa statistic. In all, 370 vaginal samples (185 self- and 185 practitioner-collected swabs) had a narrow range of values for the number of albumin gene copies and a significant correlation coefficient (Spearman's rho = 0.532; p < 0.001). The agreement between both sampling methods was excellent (Spearman's rho was 0.748 for Atopobium vaginae, 0.918 for Lactobacillus species, 0.940 for Gardnerella vaginalis; p < 0.001), especially for high concentrations of A. vaginae (≥10(8) copies/mL; kappa value = 0.973; p < 0.001) and G. vaginalis (≥10(9) copies/mL; kappa value = 0.903; p < 0.001). This study demonstrates the validity and reliability of self- versus practitioner-collected swabs for the molecular quantification of Lactobacillus species, G. vaginalis, and A. vaginae.

Taxonogenomics description of Bacillus marasmi sp. nov., a new species isolated from the stool sample.

Using the culturomics method, two strains were isolated, identified, and characterised following the taxonogenomics concept. Bacillus marasmi sp. nov. strain Marseille-P3556 (= CSURP3556) is isolated from a 13-month-old girl living in Niger. The phylogenetic tree, phenotypic criteria, and genomic analysis described here clearly show that this bacterium is different from previously known bacterial species withstanding in nomenclature and new members of Bacillus genus.

Taxonogenomics description of Arcanobacterium urinimassiliense sp. nov., a new bacterial species isolated from urine sample.

Strain Marseille-P3248т is a new species from the order Actinomycetales that was isolated from the urine sample of a girl aged 20 months with rotavirus gastroenteritis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P3248т exhibits 94.73% sequence similarity with Arcanobacterium pluranimalium strain M430/94/2, a phylogenetically related species with standing in nomenclature. Its genome size is 1 667 964 bp with 49.1% G + C content. Strain Marseille-P3248т (= CSURP3248) is the type strain of the new species Arcanobacterium urinimassiliense sp. nov.

Neobacillus massiliamazoniensis sp. nov., a new bacterial species isolated from stool sample of an inhabitant of the Amazon region.

Using a culturomics approach, a strain was isolated, identified and characterised following the taxonogenomics concept. Neobacillus massiliamazoniensis sp. nov., strain LF1T (=CSURP1359) was isolated from human stool. The 16S rRNA gene sequence analysis of strain LF1T (accession number: LK021124) exhibits 98.32% similarity levels with Neobacillus bataviensis strain IDA1115 (accession number: NR_036766.1), the phylogenetically closest related species with standing in nomenclature. The draft genome size of strain LF1T (accession number: CVRB00000000) is 4.6 Mbp with a G+C content of 34.1 mol%. Analysis of phylogenic tree, genomic analysis and phenotypic criteria described here sufficiently prove that this bacterium is different from previously known bacterial species with standing in nomenclature and represents a new Neobacillus species belonging to Firmicutes phylum.

Virgibacillus doumboii sp. nov., a halophilic bacterium isolated from the stool of a healthy child in Mali.

A moderately halophilic and strictly aerobic bacterium was isolated from a human stool as part of a study on the diagnosis of childhood malnutrition in Mali. Strain Marseille-Q1616T is a Gram-stain-positive, rod-shaped, catalase-positive and oxidase-negative bacterium. It has a genome size of 3.91 Mbp with 39.79% G+C content, which contains 3954 protein-coding genes including genes encoding phosphomycin resistance and Listeria monocytogenes, 16 rRNA genes and 64 tRNA genes. Strain Marseille-Q1616T exhibited a 96.3% 16S rRNA gene sequence similarity and shared an OrthoANI value of 70.64% (the highest observed) with Virgibacillus kekensis, the phylogenetically closest validly published species. Based on phenotypic and phylogenetic evidence and genomic average nucleotide identity values, we suggest the creation of a new species within the Virgibacillus genus, named Virgibacillus doumboii sp. nov., type strain Marseille-Q1616T (= CSURQ1616).

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

Non-contiguous finished genome sequence and description of Bartonella saheliensis sp. nov. from the blood of Gerbilliscus gambianus from Senegal.

Bartonella saheliensis strain 077 (= CSUR B644T; = DSM 28003T) is a new bacterial species isolated from blood of the rodent Gerbilliscus gambianus captured in the Sine-Saloum region of Senegal. In this work we describe the characteristics of this microorganism, as well as the complete sequence of the genome and its annotation. Its genome has 2 327 299 bp (G+C content 38.4%) and codes for 2015 proteins and 53 RNA genes.

Description of Prevotella rectalis sp. nov., a new bacterium isolated from human rectum.

Using a taxonogenomics method, we describe here a Gram-negative bacterium named Prevotella rectalis sp. nov., strain Marseille-P4334T (= CSUR P4334) isolated from the rectum. Strain Marseille-P4334T has a genome that measure 3.03 Mbp with 43.3 mol% G + C content.

Corynebacterium dentalis sp. nov., a new bacterium isolated from dental plaque of a woman with periodontitis.

Strain Marseille-P4122T is a new species from the order Corynebacteriales that was isolated from the dental plaque of a woman with periodontitis. It is a facultative anaerobic Gram-positive rod-shaped bacterium. Strain Marseille-P4122T exhibited a 98.19% sequence identity with Corynebacterium suicordis strain P81/02, the phylogenetically closely related species with standing in nomenclature. The draft genome size of strain Marseille-P4122T is 2.49 Mb with 60.1% G + C content. We propose that strain Marseille-P4122T (=CSURP4122) is the type strain of the new species Corynebacterium dentalis sp. nov.

Taxonogenomics description of Bacillus dakarensis sp. nov., Bacillus sinesaloumensis sp. nov. and Bacillus massiliogabonensis sp. nov., three new species isolated from human stools.

Using microbial culturomics, three Bacillus strains were isolated, identified and characterized following the taxonogenomics strategy. Bacillus dakarensis strain Marseille-P3515T (=CSURP3515), Bacillus sinesaloumensis strain Marseille-P3516T (=CSURP3516), and Bacillus massiliogabonensis strain Marseille-P2639T (=CSURP2639) were isolated from human stool samples. The phylogenetic analysis, phenotypic characteristics and genotypic data presented here prove that these three bacteria are different from previously known bacterial species with standing in nomenclature and represent new Bacillus species.

Detailed description of Senegalia massiliensis strain SIT17T , a bacterium isolated from the human gut.

Strain SIT17T was isolated from the stool of a healthy 13-month-old Senegalese boy. It is a Gram-positive, anaerobic, rod-shaped, non-spore-forming and mobile bacterium. It exhibited 92.74% 16S rRNA gene sequence similarity with the Brassicibacter thermophilus strain Cel2f, the phylogenetically most closely related species. Its genome is about 2.87 Mb long with 27.39 mol% G + C content. We provide more details of Senegalia massiliensis strain SIT17T (= CSURP2130 = DSM 103071), the creation of which was previously announced.

Megasphaera vaginalis sp. nov. and Anaerococcus vaginimassiliensis sp. nov., new bacteria isolated from vagina of French woman with bacterial vaginosis.

Using the culturomics method, two strains were isolated, identified and characterized following the taxonogenomics concept. Megasphaera vaginalis sp. nov. strain Marseille-P4512 (= CSURP4512) and Anaerococcus vaginimassiliensis sp. nov. strain Marseille-P4857 (= CSURP4857) were isolated from the vagina of a French woman. The phylogenic tree, phenotypic criteria and genomic analysis described here clearly show that these two bacteria are different from previously known bacterial species with standing in nomenclature and new members of Firmicutes phylum.

Two new bacteria isolated from vagina of a patient with vaginosis: Atopobium massiliense sp. nov. and Butyricimonas vaginalis sp. nov.

Two new bacterial strains, Marseille-P4126 (=CSURP4126) and Marseille-P4593 (=CSURP4593), were isolated from the vaginal sample of a French woman with vaginosis. These strains were identified and characterized using the taxonogenomics method. The findings from phylogenetic tree interpretation, phenotypic criteria and genomic analysis provided here distinctly display that Atopobium massiliense sp. nov. and Butyricimonas vaginalis sp. nov. are new members of the genus Atopobium and Butyricimonas, respectively.

Peripherally inserted central catheters: a hidden emerging cause of infection outbreaks.

In our institution, between January 2010 and December 2017, 15 140 peripherally inserted central catheters (PICCs) were inserted in 12 314 patients. Using time-series analysis to evaluate the annual historical trend (AHT), we observed a significant increase in bloodstream infections (BSIs; AHT = 24; p < 0.001) and associated deaths (AHT = 3; p 0.02) in patient with PICCs. The risk of experiencing a BSI was significantly higher in patients with PICCs (odds ratio = 9.6; 95% confidence interval, 9.08-10.18; p < 0.001). To reduce PICC-related BSIs and their related mortality, it is important to limit the overuse of PICCs and to implement a 'no PICC' policy by limiting the insertion of PICCs to situations without other available options.

Taxono-genomics and description of Gordonibacter massiliensis sp. nov., a new bacterium isolated from stool of healthy patient.

Using the taxono-genomics concept, we describe here a strictly anaerobic Gram-positive bacillus. This strain was isolated from the stool sample of a 50-year-old healthy Bedouin woman. The 16S rRNA gene sequence analysis and the whole-genome sequencing showed that this isolate belonged to the genus Gordonibacter in the family Eggerthellaceae. Based on these criteria, we propose the creation of Gordonibacter massiliensis sp. nov., strain Marseille-P2775T (= CSUR P2775).

Description of Clostridium cagae sp. nov., Clostridium rectalis sp. nov. and Hathewaya massiliensis sp. nov., new anaerobic bacteria isolated from human stool samples.

Using culturomics methods, three strains were isolated, identified and characterized following the taxonogenomics concept. Clostridium cagae strain Marseille-P4344T (=CSURP4344), Clostridium rectalis strain Marseille-P4200T (=CSURP4200) and Hathewaya massiliensis strain Marseille-P3545T (=CSURP3545) were isolated from human stool samples. The phylogenetic reconstruction, phenotypic criteria and genomic analyses were carried out and demonstrated that these three bacteria are different from previously known bacterial species with standing in nomenclature and were classified as new members of the Clostridiaceae family.