Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for Rapid Identification of Ebola Virus Disease in Heat-Treated Blood Samples Obtained in Sierra Leone and the United Kingdom.

Rapid Ebola virus (EBOV) detection is crucial for appropriate patient management and care. The performance of the FilmArray BioThreat-E test (v2.5) using whole-blood samples was evaluated in Sierra Leone and the United Kingdom and was compared with results generated by a real-time Ebola Zaire PCR reference method. Samples were tested in diagnostic laboratories upon availability, included successive samples from individual patients, and were heat treated to facilitate EBOV inactivation prior to PCR. The BioThreat-E test had a sensitivity of 84% (confidence interval [CI], 64% to 95%) and a specificity of 89% (CI, 73% to 97%) in Sierra Leone (n = 60; 44 patients) and a sensitivity of 75% (CI, 19% to 99%) and a specificity of 100% (CI, 97% to 100%) in the United Kingdom (n = 108; 70 patients) compared to the reference real-time PCR. Statistical analysis (Fisher's exact test) indicated there was no significant difference between the methods at the 99% confidence level in either country. In 9 discrepant results (5 real-time PCR positives and BioThreat-E test negatives and 4 real-time PCR negatives and BioThreat-E test positives), the majority (n = 8) were obtained from samples with an observed or probable low viral load. The FilmArray BioThreat-E test (v2.5) therefore provides an attractive option for laboratories (either in austere field settings or in countries with an advanced technological infrastructure) which do not routinely offer an EBOV diagnostic capability.

The Use of CHROMID® Colistin R for the Detection of Colistin-Resistant Gram-Negative Bacteria in Positive Blood Cultures.

The aim of this study was to assess the utility of CHROMID® Colistin R for direct detection of colistin-resistant Gram-negative bacteria from positive blood cultures. A total of 390 blood cultures from hospitalised patients containing Gram-negative bacteria were included in this study. These blood cultures were referred to clinical laboratories in the United Kingdom and Türkiye. A further 16 simulated positive blood culture bottles were included that contained a range of colistin-resistant strains as well as susceptible control strains. Fluid from each positive blood culture was diluted 1/200 in saline and 10 µL aliquots cultured onto cystine-lactose-electrolyte-deficient agar and CHROMID® Colistin R. All recovered bacteria were identified, and for Gram-negative bacteria, their minimum inhibitory concentration of colistin was measured using the broth microdilution method. From a total of 443 Gram-negative isolates, 57 colistin-resistant isolates were recovered, of which 53 (93%) grew on CHROMID® Colistin R within 18 h. Of the 377 isolates determined to be colistin-susceptible, only 9 isolates were able to grow, including 6 isolates of Pseudomonas aeruginosa. For positive blood cultures that are shown to contain Gram-negative bacteria, culture on CHROMID® Colistin R is a useful diagnostic tool to detect susceptibility or resistance to colistin within 18 h.

Aetiology of paediatric pneumonia after the introduction of pneumococcal conjugate vaccine.

We describe the aetiology of community-acquired pneumonia in children before and after the introduction of the pneumococcal conjugate vaccination (PCV) programme in 2006. Prospective studies were conducted in 2001-2002 (pre-vaccine) and 2009-2011 (post-vaccine) of children aged 0-16 years with radiologically confirmed pneumonia seen in hospital. Investigations included culture, serology, immunofluorescence antibody and urine antigen testing, with an increased use of PCR assays and expanded panels of pathogens in the post-vaccine study. 241 and 160 children were enrolled in the pre- and post-vaccine studies, respectively (73% aged <5 years). Identification of a causative pathogen was higher post-vaccination (61%) than pre-vaccination (48.5%) (p=0.019). Rates of bacterial infections were not different between post- and pre-vaccine studies (17.5% versus 24%, p=0.258). Viral (31%) and mixed (12.5%) infections were found more often post-vaccination (19.5%, p=0.021) than pre-vaccination (5%, p=0.015). Rates of identified pneumococcal infections were comparable between pre- and post-vaccine studies (14.7% versus 17.4%, p=0.557). Diagnosis of pneumococcal infection post-vaccination improved when PCR was used compared to culture (21.6% versus 6%, p=0.0004). Serotypes included in PCV13 but not PCV7 were identified in 75% (18 out of 24) post-vaccination. Infection with nonvaccine pneumococcal serotypes continues to be a significant cause of pneumonia in children in the UK.

Comparison of the Luminex NxTAG respiratory pathogen panel and a multiplex in-house real-time PCR panel for the detection of respiratory viruses in symptomatic patients.

Purpose. To evaluate the Luminex NxTAG respiratory pathogen panel (NxTAG RPP) for the detection of respiratory viruses in clinical samples from patients with the symptoms of respiratory infection.Methodology. The NxTAG RPP was compared to an in-house multiplex real-time PCR panel (LDT) for the detection of respiratory viruses in 314 clinical samples from patients with the symptoms of respiratory infection.Results. Thirty-one samples were negative in both tests and 193 samples contained a single virus that was detected in both tests. Polymicrobial infections were detected in 74 samples, with 268 samples overall having concordant results in both assays, and there were a total of 51 discordant results in 44 samples. Two samples were invalid in the NxTAG RPP assay and were excluded from the final analysis. The overall agreement between the NxTAG RPP and LDT was very high, as indicated by the Kappa coefficients, which ranged from 0.85 for metapneumovirus up to 0.96 for RSV A, and the overall percentage agreement values of 96.2 % for enterovirus/rhinovirus and 100 % for influenza A, influenza B, PIV 4 and RSV B.Conclusion. The NxTAG RPP is a sensitive and specific test for the detection of respiratory viruses and the high sample throughput and low hands-on time make the NxTAG RPP assay suitable for screening clinical samples for respiratory pathogens.

Cavitatory lung disease complicating empyema in children.

OBJECTIVE: The incidence of empyema has increased dramatically in children in the UK over the last decade. Streptococcus pneumoniae (S. pneumoniae) serotype 1 is the dominant serotype. We have observed more pneumatocoele and bronchopleural fistulae formation over this time. AIM: Our aim was to determine the number of children who developed cavitatory disease as a complication of empyema at a tertiary referral centre and whether there was any association with S. pneumoniae serotype 1. METHOD: We reviewed 75 cases presenting with empyema or parapneumonic effusion between February 1997 and July 2003. Bacterial culture and pneumococcal antigen detection were supplemented by real-time polymerase chain reaction (PCR) to detect pneumococcal DNA. RESULTS: Cavitatory disease was present in 15 cases. Three children developed bronchopleural fistulae. S. pneumoniae was detected in 13 of 15 cases (4 cases serotype 1, 3 serotype 3, 2 serotype 14, and 2 serotype 9V; serotype assay was not performed in two cases). Staphylococcus aureus (S. aureus) was isolated in one case. No organism was isolated in the final case but an Antistreptolysin-O titre was >800 U/ml on two occasions suggestive of group A streptococcal infection. CONCLUSION: Twenty percentage of cases of empyema in our series were complicated by cavitatory lung disease. It is an important complication of childhood empyema associated classically with S. aureus, but these data suggest that S. pneumoniae now appears to be the main cause. There does not seem to be an association with any particular serotype.

Rapid detection and quantification of CMV DNA in urine using LightCycler-based real-time PCR.

A real-time quantitative PCR-hybridisation assay was developed for the detection of human cytomegalovirus DNA in clinical material. The assay is based on a LightCycler (LC) and provides both rapid results (<1 h) and quantification over a broad dynamic range (2 x 10(3)-5 x 10(8) CMV DNA copies/ml). Given that the assay showed a 3-fold increase in sensitivity compared to detection of early antigen fluorescent foci (DEAFF) testing of urine samples, we investigated the practicality of testing surveillance such specimens from immunocompromised patients at risk of CMV disease. Over a 12-month period, CMV DNA was detected in 81 (7%) of 1154 urine samples examined. A total of 28 patients tested positive; urine viral loads were higher in 13 infants being investigated for suspected congenital infection (median 1.6 x 10(5) copies/ml) compared with 15 transplant recipients (median 9 x 10(3) copies/ml). Urine samples could be tested directly without processing such that results were available in <1h. Real-time PCR provided information on the quantification of CMV DNA in urine and proved a reliable method for the surveillance of immunocompromised patients at risk of CMV disease. This approach should facilitate a better understanding of the epidemiology and natural history of CMV disease. Moreover, LC-based quantitative PCR is a potentially valuable tool for the management of CMV disease; assisting with the prompt initiation of treatment and assessing therapeutic response.

Detection of pneumolysin in sputum.

Western blot detection of the species-specific pneumococcal product, pneumolysin (SPN), was shown to be almost as sensitive as PCR for the non-cultural detection of pneumococci in 27 Streptococcus pneumoniae culture-positive sputa from patients stated to have chest infections. Both techniques were considerably more sensitive than counter-current immuno-electrophoresis for pneumococcal capsular polysaccharide antigens (CPS-CIE) on the same specimens. Sensitivities for PCR, SPN-immunoblotting and CPS-CIE were 100%, 85% and 67%, respectively. In 11 S. pneumoniae culture-negative sputa taken from patients receiving antibiotics, but with proven recent pneumococcal infection, PCR and SPN-blot were positive in six (in two of which CPS-CIE was also positive), PCR alone was positive in one and SPN-blot alone was positive in one. In 11 S. pneumoniae culture-negative samples from patients not receiving antibiotics, all three tests were negative in eight, PCR was positive in three (in one of which CPS-CIE was also positive), but SPN-blot was negative in all 11. In 16 S. pneumoniae culture-negative samples from patients receiving antibiotics and with no known recent pneumococcal infections, one or more non-cultural test was positive in 11. Although further evaluation is required to assess the significance of pneumolysin detection in relation to carriage and infection and to devise a more suitable test format, these preliminary studies suggest that pneumolysin detection is a promising new approach to the non-cultural diagnosis of pneumococcal chest infection.

Pneumococcal diagnosis and serotypes in childhood community-acquired pneumonia.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced routinely in the UK from September 2006 and replaced by PCV13 in 2010. In a prospective study from 2009 to 2011 of 160 children aged ≤16 years with radiologically confirmed pneumonia, likely pneumococcal infections were identified in 26%. Detection of pneumococci was improved with polymerase chain reaction compared to culture (21.6% versus 6% of children tested, P = 0.0004). Where serotyping was possible, all (n = 23) were non-PCV7 but PCV13 serotypes; 1 (43.5%), 3 (21.7%), 7A/F, and 19A (17.4% each).

Evaluation of the Cepheid respiratory syncytial virus and influenza virus A/B real-time PCR analyte specific reagent.

Two rapid real-time RT-PCR assays, specific for respiratory syncytial virus (RSV) and influenza A and B, were evaluated for the detection of these viruses in clinical respiratory samples. The RSV assay was applied to 100 samples and the Influenza A and B assay applied to 96 samples all of which had been tested previously by an "in-house" multiplex real-time PCR assay. Forty-three samples were negative for RSV by both methods and 56 samples were positive by both methods. One sample was negative by the new RSV assay although it was positive for RSV A by the "in-house" test. Thirty-nine samples were negative for influenza virus by both methods and 55 samples were positive by both assays. Two samples were negative by the new influenza assay however they were positive by the "in-house" influenza assay. The new assays did not cross react with samples containing other viruses including parainfluenza 1, 2, and 4; human metapnuemovirus; coronavirus 229E, NL63, OC43; rhinovirus; adenovirus; bocavirus and had a specificity of 100% and a sensitivity of 98.2% for RSV and 96.5% for influenza respectively. The results of this study demonstrate that the new assays are specific and sensitive for the detection of RSV and influenza viruses in clinical samples.