ABSTRACT
The Sequenom MassARRAY iPLEX single-nucleotide polymorphism (SNP) typing platform uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with single-base extension PCR for high-throughput multiplex SNP detection. In this study, we investigated the use of iPLEX MassARRAY technology for methicillin-resistant Staphylococcus aureus (MRSA) genotyping. A 16-plex MassARRAY iPLEX GOLD assay (MRSA-iPLEX) was developed that targets a set of informative SNPs and binary genes for MRSA characterization. The method was evaluated with 147 MRSA isolates, and the results were compared with those of an established SYBR Green-based real-time PCR system utilizing the same SNP-binary markers. A total of 2352 markers belonging to 44 SNP-binary profiles were analysed by both real-time PCR and MRSA-iPLEX. With real-time PCR as the reference standard, MRSA-iPLEX correctly assigned 2298 of the 2352 (97.7%) markers. Sequence variation in the MRSA-iPLEX primer targets accounted for the majority of MRSA-iPLEX erroneous results, highlighting the importance of primer target selection. MRSA-iPLEX provided optimal throughput for MRSA genotyping, and was, on a reagent basis, more cost-effective than the real-time PCR methods. The 16-plex MRSA-iPLEX is a suitable alternative to SYBR Green-based real-time PCR typing of major sequence types and clonal complexes of MRSA.
Subject(s)
Methicillin-Resistant Staphylococcus aureus/classification , Methicillin-Resistant Staphylococcus aureus/genetics , Molecular Typing/methods , Polymorphism, Single Nucleotide , Real-Time Polymerase Chain Reaction/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Staphylococcal Infections/microbiology , Costs and Cost Analysis , Humans , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Real-Time Polymerase Chain Reaction/economics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/economicsABSTRACT
Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.
Subject(s)
Cystic Fibrosis/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/genetics , Real-Time Polymerase Chain Reaction/methods , Australia , Base Sequence , Electrophoresis, Gel, Pulsed-Field , Humans , Molecular Sequence Data , Polymorphism, Single Nucleotide , Pseudomonas aeruginosa/isolation & purificationABSTRACT
Since the role of respiratory viruses in lung exacerbations of patients with cystic fibrosis has been hampered by the difficulty of detecting viruses in viscous sputum specimens, a multiplex reverse transcriptase PCR (RT-PCR) assay combined with colorimetric amplicon detection was tested for the identification of seven common respiratory viruses in the sputa of cystic fibrosis patients. Of 52 sputa from 38 patients, 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for influenza B, 3 for parainfluenza 1, 3 for influenza A and 2 for respiratory syncytial virus). These results suggest that the RT-PCR method carried out on sputum may provide a convenient means of investigating the role of virus infection in lung exacerbations of cystic fibrosis patients.
Subject(s)
Cystic Fibrosis/virology , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Viruses/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Sputum/virology , Adolescent , Adult , Case-Control Studies , Cystic Fibrosis/diagnosis , Female , Humans , Incidence , Male , Probability , Prognosis , RNA, Viral/analysis , Respiratory Function Tests , Respiratory Syncytial Virus Infections/epidemiology , Sensitivity and Specificity , Severity of Illness IndexABSTRACT
Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious. Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded. The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance. In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study.
Subject(s)
Anti-Bacterial Agents/pharmacology , Cystic Fibrosis/epidemiology , Cystic Fibrosis/microbiology , Pseudomonas Infections/diagnosis , Pseudomonas Infections/epidemiology , Pseudomonas aeruginosa/classification , Adolescent , Adult , Age Distribution , Australia/epidemiology , Child , Confidence Intervals , Cross Infection/prevention & control , Cystic Fibrosis/diagnosis , Drug Resistance, Bacterial , Electrophoresis, Gel, Pulsed-Field , Female , Hospital Units , Humans , Incidence , Male , Microbial Sensitivity Tests , Probability , Prognosis , Prospective Studies , Pseudomonas aeruginosa/drug effects , Risk Assessment , Sex Distribution , Sputum/microbiology , Statistics, NonparametricABSTRACT
Three Herpes Simplex Virus LightCycler polymerase chain reaction assays were compared for the detection of herpes simplex virus in 48 swab specimens. The assays comprised of one in-house assay and two commercial kits: the Artus HSV LC RealArt PCR kit and the Roche LightCycler HSV 1/2 Detection kit. On the whole, the three assays had comparable sensitivities. However, differentiation of herpes simplex virus types 1 and 2 by melting curve analysis was problematic in all assays. Overall, the results highlight the limitations of typing herpes simplex virus by melting curve analysis.
