The current landscape of nucleic acid tests for filovirus detection.

Nucleic acid testing (NAT) for pathogenic filoviruses plays a key role in surveillance and to control the spread of infection. As they share clinical features with other pathogens, the initial spread of these viruses can be misdiagnosed. Tests that can identify a pathogen in the initial stages of infection are essential to control outbreaks. Since the Ebola virus disease (EVD) outbreak in 2014-2016 several tests have been developed that are faster than previous tests and more suited for field use. Furthermore, the ability to test for a range of pathogens simultaneously has been expanded to improve clinical pathway management of febrile syndromes. This review provides an overview of these novel diagnostic tests.

Dental and microbiological risk factors for hospital-acquired pneumonia in non-ventilated older patients.

METHODS: We obtained a time series of tongue/throat swabs from 90 patients with lower limb fracture, aged 65-101 in a general hospital in the North East of England between April 2009-July 2010. We used novel real-time multiplex PCR assays to detect S. aureus, MRSA, E. coli, P. aeruginosa, S. pneumoniae, H. influenza and Acinetobacter spp. We collected data on dental/denture plaque (modified Quigley-Hein index) and outcomes of clinician-diagnosed HAP. RESULTS: The crude incidence of HAP was 10% (n = 90), with mortality of 80% at 90 days post discharge. 50% of cases occurred within the first 25 days. HAP was not associated with being dentate, tooth number, or heavy dental/denture plaque. HAP was associated with prior oral carriage with E. coli/S. aureus/P.aeruginosa/MRSA (p = 0.002, OR 9.48 95% CI 2.28-38.78). The incidence of HAP in those with carriage was 35% (4% without), with relative risk 6.44 (95% CI 2.04-20.34, p = 0.002). HAP was associated with increased length of stay (Fishers exact test, p=0.01), with mean 30 excess days (range -11.5-115). Target organisms were first detected within 72 hours of admission in 90% participants, but HAP was significantly associated with S. aureus/MRSA/P. aeruginosa/E. coli being detected at days 5 (OR 4.39, 95%CI1.73-11.16) or 14 (OR 6.69, 95%CI 2.40-18.60). CONCLUSIONS: Patients with lower limb fracture who were colonised orally with E. coli/ S. aureus/MRSA/P. aeruginosa after 5 days in hospital were at significantly greater risk of HAP (p = 0.002).

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.

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.

Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni.

Multilocus sequence typing (MLST) has been proven useful for the study of the global population structure of Campylobacter jejuni; however, its usefulness for the investigation of outbreaks of disease caused by C. jejuni has not been proven. In this study, MLST plus sequencing of the flaA short variable region (SVR) were applied to 47 isolates from 12 outbreaks of C. jejuni infection whose relatedness has been determined previously, and the results were compared to those of serotyping and pulsed-field gel electrophoresis (PFGE). Isolates implicated in an outbreak were indistinguishable by all four subtyping methods, with sporadic isolates being distinguished from outbreak isolates. Two sporadic isolates from one outbreak were resistant to SmaI digestion and therefore nontypeable by PFGE but were differentiated from the outbreak strain by the other methods. PFGE and flaA SVR typing were the most discriminatory methods, with discriminatory indices (DI) of 0.930 and 0.923, respectively. However, an epidemic strain from one outbreak was distinguished from the other outbreak isolates by flaA SVR typing; its flaA allele was different at five nucleotides, suggesting that this change was possibly mediated by recombination. MLST was less discriminatory than PFGE and flaA SVR typing (DI = 0.859), and many of the epidemic strains possessed common sequence types (STs) including ST-8, -21, -22, and -42. However, further discrimination within STs was achieved by flaA SVR typing or PFGE. The results from this study demonstrate that a combined approach of MLST plus flaA SVR typing provides a level of discrimination equivalent to PFGE for outbreak investigations.

Clonal complexes of Campylobacter jejuni identified by multilocus sequence typing correlate with strain associations identified by multilocus enzyme electrophoresis.

Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) with SmaI were used to subtype 55 isolates of Campylobacter jejuni from a diverse range of human and animal sources previously characterized by multilocus enzyme electrophoresis (MEE). MEE and MLST targeted 11 and 7 loci, respectively, and all loci were unique to each method. MEE, MLST, and PFGE identified 40, 37, and 48 discrete subtypes, respectively, with many of the subtypes occurring only once within the data set. Simpson's indices of diversity were calculated to be 0.979, 0.966, and 0.994 for MEE, MLST, and PFGE, respectively, demonstrating that MEE and MLST had similar discriminatory powers but that PFGE was more discriminatory. Allele diversity was higher in the MLST loci; individual single-locus diversities for the 11 MEE loci and the 7 MLST loci were 0.491 and 0.854, respectively. The clonal complexes recognized by MLST correlated with the strain associations previously recognized by MEE and contained some isolates indistinguishable by PFGE. Many clusters contained isolates from diverse geographical regions and from both humans and animals. These results demonstrate the usefulness of MLST for investigation of the global epidemiology of this important pathogen and illustrate its potential to identify indistinguishable strains or clones in geographically distinct regions.

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture.

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.