Rapid PCR detection of group a streptococcus from flocked throat swabs: A retrospective clinical study

<p>Abstract</p> <p>Background</p> <p>Rapid diagnosis of GAS pharyngitis may improve patient care by ensuring that patients with GAS pharyngitis are treated quickly and also avoiding unnecessary use of antibiotics in those without GAS infection. Very few molecular methods for detection of GAS in clinical throat swab specimens have been described.</p> <p>Methods</p> <p>We performed a study of a laboratory-developed internally-controlled rapid Group A streptococcus (GAS) PCR assay using flocked swab throat specimens. We compared the GAS PCR assay to GAS culture results using a collection of archived throat swab samples obtained during a study comparing the performance of conventional and flocked throat swabs.</p> <p>Results</p> <p>The sensitivity of the GAS PCR assay as compared to the reference standard was 96.0% (95% CI 90.1% to 98.4%), specificity 98.6% (95% CI 95.8% to 99.5%), positive predictive value (PPV) 96.9% (95% CI 91.4% to 99.0%) and negative predictive value (NPV) of 98.1% (95% CI 95.2% to 99.2%). For conventional swab cultures, sensitivity was 96.0% (95% CI 90.1% to 98.4%), specificity 100% (95% CI 98.2% to 100%), PPV 100%, (95% CI 96.1% to 100%) and NPV 98.1% (95% CI 95.2% to 99.3%)</p> <p>Conclusions</p> <p>In this retrospective study, the GAS PCR assay appeared to perform as well as conventional throat swab culture, the current standard of practice. Since the GAS PCR assay, including DNA extraction, can be performed in approximately 1 hour, prospective studies of this assay are warranted to evaluate the clinical impact of the assay on management of patients with pharyngitis.</p

Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel

Background: Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. Methods: RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. Results: One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P\u3c0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. Conclusion: The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship

Suboptimal clinical response to ciprofloxacin in patients with enteric fever due to Salmonella spp. with reduced fluoroquinolone susceptibility: a case series

BACKGROUND: Salmonella spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases. METHODS: Salmonella enterica Serotype Typhi (S. Typhi) and Serotype Paratyphi (S. Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed. RESULTS: Seven of 21 (33%) S. Typhi and S. Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L). Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization. CONCLUSIONS: Our cases provide further evidence that reduced fluoroquinolone susceptibility of S. Typhi and S. Paratyphi is clinically significant. Laboratories should test extra-intestinal Salmonella spp. for reduced fluoroquinolone susceptibility

Cloxacillin versus vancomycin for presumed late-onset sepsis in the Neonatal Intensive Care Unit and the impact upon outcome of coagulase negative staphylococcal bacteremia: a retrospective cohort study

BACKGROUND: Coagulase negative staphylococcus (CONS) is the main cause of late-onset sepsis in Neonatal Intensive Care Units (NICU). Although CONS rarely causes fulminant sepsis, vancomycin is frequently used as empiric therapy. Indiscriminate use of vancomycin has been linked to the emergence of vancomycin resistant organisms. The objective of this study was to compare duration of CONS sepsis and mortality before and after implementation of a policy of selective vancomycin use and compare use of vancomycin between the 2 time periods. METHODS: A retrospective study was conducted of infants ≥4 days old, experiencing signs of sepsis with a first positive blood culture for CONS, during two 12-month periods. Late-onset sepsis was treated empirically with vancomycin and gentamicin during period 1, and cloxacillin and gentamicin during period 2. The confidence interval method was used to assess non-inferiority of the outcomes between the two study groups. RESULTS: There were 45 episodes of CONS sepsis during period 1 and 37 during period 2. Duration of sepsis was similar between periods (hazard ratio of 1.00, 95%CI: 0.64, 1.57). One death during period 2 was possibly related to CONS sepsis versus none in period 1. Vancomycin was used in 97.8% of episodes in period 1 versus 81.1% of episodes in period 2. CONCLUSION: Although we failed to show non-inferiority of duration of sepsis in the cloxacillin and gentamicin group compared to the vancomycin and gentamicin group, duration of sepsis was clinically similar. Restricting vancomycin for confirmed cases of CONS sepsis resistant to oxacillin appears effective and safe, and significantly reduces vancomycin use in the NICU

Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNγ-like response

MN and CP were supported by the Wellcome Trust (www.wellcome.ac.uk) Institutional Strategic Support Fund and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1; www.dfg.de).The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication.Publisher PDFPeer reviewe