ABSTRACT
BACKGROUND: The spread of carbapenemase-producing Enterobacterales (CPE) is a global health problem. Gastrointestinal tract carriage makes faeces or rectal swabs the recommended screening methods. AIM: To assess the impact of three laboratory screening strategies for CPE on positivity rates and infection prevention and control in a hospital setting in North West England from 2015 to 2017. METHODS: In a retrospective study, time to CPE-positive and -negative results, number of new CPE-positive patients identified, and number of hospital bed-days lost/wards affected were measured for each of three CPE screening strategies; culture plus phenotypic tests, culture plus polymerase chain reaction (PCR), and PCR only (phases 1, 2 and 3, respectively). FINDINGS: The fastest time to CPE results was PCR only (median: 4.0 h), then culture plus PCR (median: 47.6 h), then culture plus phenotypic tests (median: 49.8 h) (P < 0.001). The mean numbers of hospital bed-days lost per month decreased between phases 2 and 3 (P = 0.01). The mean number of wards/units affected by CPE increased from phase 1 (2.57) to phase 2 (7.71), then decreased in phase 3 (3.86). The percentage positivity rate for phases 1, 2, and 3 were 2.01, 1.38, and 1.55 respectively. From May to October, the number of new CPE-positive patients was lower for phases 1 and 3 than for phase 2. During all three phases there was a peak in the number of newly identified CPE carriers in August. CONCLUSION: This study provides evidence that using a rapid PCR to screen rectal or faeces swabs enables more timely infection prevention and control measures when compared with culture-based methods. A reduction in bed-days lost due to CPE was observed when rapid molecular screening was introduced.
ABSTRACT
BACKGROUND: The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established. OBJECTIVES: To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease. STUDY DESIGN: Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated. RESULTS: Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged<15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively. CONCLUSIONS: This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC.
Subject(s)
Polyomavirus Infections/epidemiology , Polyomavirus/isolation & purification , Respiratory Tract Infections/epidemiology , Adolescent , Adult , Age Distribution , Aged , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Polymerase Chain Reaction , Polyomavirus/genetics , Polyomavirus Infections/virology , Prevalence , Respiratory Tract Infections/virology , Sequence Analysis, DNAABSTRACT
A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.
Subject(s)
ATP-Binding Cassette Transporters , Bacteremia/microbiology , DNA-Binding Proteins , Haemophilus influenzae/isolation & purification , Meningitis, Bacterial/microbiology , Neisseria meningitidis/isolation & purification , Streptococcus pneumoniae/isolation & purification , Transcription Factors , Bacteremia/diagnosis , Bacterial Proteins/genetics , Culture Media , DNA Primers , DNA, Bacterial/blood , DNA, Bacterial/cerebrospinal fluid , Haemophilus influenzae/genetics , Humans , Meningitis, Bacterial/diagnosis , Meningitis, Haemophilus/diagnosis , Meningitis, Haemophilus/microbiology , Meningitis, Meningococcal/diagnosis , Meningitis, Meningococcal/microbiology , Meningitis, Pneumococcal/diagnosis , Meningitis, Pneumococcal/microbiology , Molecular Sequence Data , Neisseria meningitidis/genetics , Polymerase Chain Reaction/methods , Sensitivity and Specificity , Streptococcus pneumoniae/genetics , Streptolysins/genetics , Taq Polymerase/metabolismABSTRACT
A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.
