Development of a real-time PCR for the specific detection of Waddlia chondrophila in clinical samples

Waddlia chondrophila is considered as an emerging human pathogen likely involved in miscarriage and lower respiratory tract infections. Given the low sensitivity of cell culture to recover such an obligate intracellular bacteria, molecular-based diagnostic approaches are warranted. We thus developed a real-time PCR that amplifies Waddlia chondrophila DNA. Specific primers and probe were selected to target the 16S rRNA gene. The PCR specifically amplified W. chondrophila but did not amplify other related-bacteria such as Parachlamydia acanthamoebae, Simkania negevensis and Chlamydia pneumoniae. The PCR exhibited a good intra-run and inter-run reproducibility and a sensitivity of less than ten copies of the positive control. This real-time PCR was then applied to 32 nasopharyngeal aspirates taken from children with bronchiolitis not due to respiratory syncytial virus (RSV). Three samples revealed to be Waddlia positive, suggesting a possible role of this Chlamydia-related bacteria in this settin

Individualized Catheter Surveillance among Neonates: A Prospective, 8-Year, Single-Center Experience

Objective. To monitor trends in central line-associated bloodstream infections and clinical sepsis (CLABICS) among neonates and to determine risk factors for infection, especially dwell time. Design. Prospective, single-center cohort study conducted from 2001 through 2008. Setting. University-affiliated tertiary care center. Methods. Individualized surveillance of catheter use and CLABICS episodes was conducted. Data were obtained via regular on-site visits made 3 times a week. Trends over time were estimated by Poisson regression, and risk factor analysis was conducted using a Cox proportional hazards model and logistic regression. Results. In all, 1,124 neonates were exposed to 2,210 central lines for a total of 12,746 catheter-days and 11,467 catheter-days at risk. The median duration of catheter use was 8 (interquartile range, 5-11) days for peripherally inserted central catheters (PICCs) and 4 (interquartile range, 2-6) days for umbilical catheters; 102 CLABICS episodes were detected. The median time to infection was 7 days. Incidence densities were 8.5 CLABICS episodes per 1,000 catheter-days at risk and 8.0 CLABICS episodes per 1,000 catheter-days. The highest rates were identified among neonates weighing 750 g or lower (14.9 CLABICS episodes per 1,000 catheter days at risk) and for PICCs (13.2 CLABICS episodes per 1,000 catheter days at risk). Catheter dwell time was associated with CLABICS for all umbilical catheters (odds ratio [OR], 1.2 per day of use [95% confidence interval {CI}, 1.1-1.3] P< .001) and for PICCs for up to 7 days (OR, 1.2 [95% CI, 1.1-1.4]; P = .041), but not thereafter (OR, 1.0 [95% CI, 0.9-1.1]; P = .90). Conclusion. Catheter dwell time is a risk factor for CLABICS during the first 7 days, irrespective of catheter type. After 7 days, PICCs are less likely to become infecte

Prevalence and diversity of Chlamydiales and other amoeba-resisting bacteria in domestic drinking water systems.

A growing number of human infections incriminate environmental bacteria that have evolved virulent mechanisms to resist amoebae and use them as a replicative niche. These bacteria are designated amoeba-resisting bacteria (ARB). Despite the isolation of these ARB in various human clinical samples, the possible source of infection remains undetermined in most cases. However, it is known that the ARB Legionella pneumophila, for instance, causes a respiratory infection in susceptible hosts after inhalation of contaminated water aerosols from various sources. The Chlamydiales order contains many ARB, such as Parachlamydia acanthamoebae or Simkania negevensis, previously implicated in human respiratory infections with no identified contamination sources. We thus investigated whether domestic water systems are a potential source of transmission of these Chlamydiales to humans by using amoebal culture and molecular methods. Other important ARB such as mycobacteria and Legionella were also investigated, as were their possible amoebal hosts. This work reports for the first time a very high prevalence and diversity of Chlamydiales in drinking water, being detected in 35 (72.9%) of 48 investigated domestic water systems, with members of the Parachlamydiaceae family being dominantly detected. Furthermore, various Legionella and mycobacteria species were also recovered, some species of which are known to be causal agents of human infections

Zoonotic Potential of Rotavirus From Swine and Bovine in South of Taiwan

Rotavirus was recognized as the virus that responsible for causing acute gastroenteritis, especially young livestock. Taiwan Center for Disease Control (CDC) confirms the majority cases of acute gastroenteritis in Taiwan on February 2015 were caused by rotavirus. In this study, we report the incidence and zoonotic impact of rotavirus strain from Taiwan. This study examined 90 (swine) and 60 (bovine) fecal samples collected from south of Taiwan in March 2015. Detection of rotavirus using VP6 gene by RT-PCR technique with amplicons 379 bp. Zoonotic potential analysis based on nucleotide sequence and phylogenetic analysis. RT-PCR utilizing the primers specific for VP6 gene detected rotavirus with positive reactions 3/30 (10%) in piglets and 1/20 (5%) in the calf. Based on the nucleotide sequences and phylogenetic analysis indicated that 1 of 3 wild strains from swine rotavirus had 85.0% - 91.1% and 1 wild strain from bovine had 78.7% - 85.9% identity relations with human strains. These findings indicated that the wild strains of swine and bovine rotavirus may broadly spread and contribute to zoonotic transmission

Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family

Comparison of clinical presentation of respiratory tract infections in H1N1/09-positive and H1N1/09-negative patients

The true burden of influenza in children is difficult to assess and is probably underestimated as clinical signs are usually nonspecific, and formal viral identification is rarely searched. In this study, we compare the clinical features of infections related to the new H1N1/09 influenza virus with infections due to other respiratory viruses in children consulting in a tertiary care pediatric hospital in Geneva. Between October 1, 2009 and February 10, 2010, 109 patients were recruited, with a median of age of 7years (range 0.1-18). There were 75 H1N1/09-positive patients (69%), and 32 (43%) had identified risk factors such as asthma or a history of wheezing. Fever (87%), cough (92%), and rhinitis (85%) were the most frequent reported presenting symptoms in both patient groups. H1N1/09-positive patients were significantly older (median of 8.2 vs. 4.6years) and were more likely to have risk factors (43% vs. 24%) and myalgias (41% vs. 20%). H1N1/09-negative patients had more wheezing episodes (29% vs. 9%), higher rates of dyspnea (28% vs. 20%) and of hospital admissions (35% vs. 16%). Conclusion: Clinical signs cannot reliably differentiate H1N1/09-positive and H1N1/09-negative patients, although we found a higher proportion of myalgias in H1N1/09-positive patients. Severity of disease was lower in H1N1/09-positive than in H1N1/09-negative patients, mostly because of a higher proportion of asthma/wheezing episodes among H1N1/09-negative patient

Comparative analysis of viral shedding in pediatric and adult subjects with central nervous system-associated enterovirus infections from 2013 to 2015 in Switzerland.

Several enterovirus (EV) genotypes can result in aseptic meningitis, but their routes of access to the central nervous system remain to be elucidated and may differ between the pediatric and adult populations. To assess the pattern of viral shedding in pediatric and adult subjects with acute EV meningitis and to generate EV surveillance data for Switzerland. All pediatric and adult subjects admitted to the University Hospitals of Geneva with a diagnosis of EV meningitis between 2013 and 2015 were enrolled. A quantitative EV real-time reverse transcriptase (rRT)-PCR was performed on the cerebrospinal fluid (CSF), blood, stool, urine and respiratory specimens to assess viral shedding and provide a comparative analysis of pediatric and adult populations. EV genotyping was systematically performed. EV positivity rates differed significantly between pediatric and adult subjects; 62.5% of pediatric cases (no adult case) were EV-positive in stool and blood for subjects for whom these samples were all collected. Similarly, the EV viral load in blood was significantly higher in pediatric subjects. Blood C-reactive protein levels were lower and the number of leucocytes/mm3 in the CSF were higher in non-viremic than in viremic pediatric subjects, respectively. A greater diversity of EV genotypes was observed in pediatric cases, with a predominance of echovirus 30 in children ≥3 years old and adults. In contrast to adults, EV-disseminated infections are predominant in pediatric subjects and show different patterns of EV viral shedding. This observation may be useful for clinicians and contribute to modify current practices of patient care

Dynamics of Bacterial Hand Contamination During Routine Neonatal Care

Abstract Objective: To evaluate the dynamics of bacterial contamination of healthcare workers' (HCWs) hands during neonatal care. Setting: The 20-bed neonatal unit of a large acute care teaching hospital in Geneva, Switzerland. Methods: Structured observation sessions were conducted. A sequence of care began when the HCW performed hand hygiene and ended when the activity changed or hand hygiene was performed again. Alcohol-based handrub was the standard procedure for hand hygiene. An imprint of the five fingertips of the dominant hand was obtained before and after hand hygiene and at the end of a sequence of care. Regression methods were used to model the final bacterial count according to the type and duration of care and the use of gloves. Results: One hundred forty-nine sequences of care were observed. Commensal skin flora comprised 72.4% of all culture-positive specimens (n = 360). Other microorganisms identified were Enterobacteriaceae (n = 55, 13.8%); Staphylococcus aureus (n = 10, 2.5%); and fungi (n = 7,1.8%). Skin contact, respiratory care, and diaper change were independently associated with an increased bacterial count; the use of gloves did not fully protect HCWs' hands from bacterial contamination. Conclusions: These data confirm that hands become progressively contaminated with commensal flora and potential pathogens during neonatal care, and identify activities at higher risk for hand contamination. They also reinforce the need for hand hygiene after a sequence of care, before starting a different task, and after glove remova

Influence of age, social patterns and nasopharyngeal carriage on antibodies to three conserved pneumococcal surface proteins (PhtD, PcpA and PrtA) in healthy young children

The acquisition of specific antibodies is paramount to protect children against pneumococcal diseases, and a better understanding of how age, ethnicity and/or Streptococcus pneumoniae (Spn) nasopharyngeal carriage influence the acquisition of antibodies to pneumococcal surface proteins (PSP) is important for the development of novel serodiagnostic and immunisation strategies. IgG antibody titres against three conserved PSP (PhtD, PcpA and PrtA) in the sera of 451 healthy children aged 1 to 24months from Israel [Jewish (50.1%) and Bedouin (49.9%)] were measured by enzyme-linked immunosorbent assay (ELISA), while nasopharyngeal swabs from these children were assessed for the presence of Spn. Globally, anti-PhtD and anti-PrtA geometric mean concentrations (GMC; EU/ml) were high at <2.5months of age [PhtD: 35.3, 95% confidence interval (CI) 30.6-40.6; PrtA: 71.2, 95 % CI 60-84.5], was lower at 5-7months of age (PhtD: 10, 95 % CI 8-12.4; PrtA: 17.9, 95 % CI 14.4-22.1) and only increased after 11months of age. In contrast, an increase in anti-PcpA was observed at 5-7months of age. Anti-PcpA and anti-PrtA, but not anti-PhtD, were significantly higher in Bedouin children (PcpA: 361.6 vs. 226.3, p = 0.02; PrtA: 67.2 vs. 29.5, p < 0.001) in whom Spn nasopharyngeal carriage was identified earlier (60% vs. 38% of carriers <6months of age, p = 0.002). Spn carriage was associated with significantly higher anti-PSP concentrations in carriers than in non-carriers (p < 0.001 for each PSP). Thus, age, ethnicity and, essentially, nasopharyngeal carriage exert distinct cumulative influences on infant responses to PSP. These specific characteristics are worthwhile to include in the evaluation of pneumococcal seroresponses and the development of new PSP-based vaccine

Hand Hygiene and Glove Use Behavior in an Italian Hospital

In an Italian hospital, we observed that hand hygiene was performed in 638 (19.6%) of 3,253 opportunities, whereas gloves were worn in 538 (44.2%) 1,218 of opportunities. We observed an inverse correlation between the intensity of care and the rate of hand hygiene compliance (R2=0.057; P<.001), but no such association was observed for the rate of glove use compliance (R2 = 0.014; P = .078). Rates of compliance with hand hygiene and glove use recommendations follow different behavioral pattern