What assay is optimal for the diagnosis of measles virus infection? An evaluation of the performance of a measles virus real-time reverse transcriptase PCR using the Cepheid SmartCycler® and antigen detection by immunofluorescence
Section snippets
Background
In March 2014, the World Health Organization (WHO) announced that measles elimination had been achieved in Australia [22]. However, the importation of measles by foreign visitors or returned travelers from areas of high prevalence continues to result in multiple localized outbreaks [3], [11], [18], [19]. Laboratory confirmation is essential to accurately monitor the epidemiology of measles and to implement effective control strategies. Detection of measles virus-specific IgM antibody on
Objectives
To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the SmartCycler® platform and measles antigen detection by immunofluorescence for the detection of measles virus. Each assay was compared against the LightCycler NAT as the reference assay.
Samples used to determine the analytical sensitivity and specificity of the SmartCycler NAT
For analysis of the lower limit of detection (LoD) of the SmartCycler assay, RNA extracted from 5 μL of the M–M–R II vaccine (CSL Limited/Merck & Co., Inc., Parkville, Victoria, Australia). Each 0.5 mL vial of M–M–R II vaccine contains ≥103 50% tissue culture infectious dose (TCID50) of the Edmonston strain of measles [5]. A solitary vial was tested, and the amount of RNA extracted from the vaccine was not quantified. Serial dilutions (neat to 10−7) of RNA extracted were then used as template
Limit of detection of measles virus and analytical specificity on the SmartCycler RT-PCR
The mean cycle threshold (Ct) values of RNA extracted from the M–M–R II vaccine tested neat, 10−1, 10−2, 10−3 and 10−4 was 22.7, 26.3, 30.3, 34.3 and 40.9, respectively. For clinical samples tested, the Ct values were noted to be marginally higher on the SmartCycler compared to the LightCycler with a mean difference of 0.89 cycles (range −1.88 to 3.38). No PCR products were obtained when nucleic acid extracts of other non-measles viruses were tested on both the SmartCycler and LightCycler
Discussion
In the present study, the SmartCycler NAT performed comparably with the reference LightCycler RT-PCR to detect measles virus in a range of clinical samples. Additional advantages of the SmartCycler NAT platform include random access and a modest level of training required for usage and interpretation of results.
One patient without clinical measles infection had measles RNA detected by RT-PCR in NTS due to recent vaccination with a measles-containing vaccine. Our diagnostic RT-PCR does not
Funding
None.
Conflict of interest
None.
Ethical approval
The investigation of individual cases of measles infection was conducted as part of public health investigations of suspected or confirmed cases of measles notified under the legal authority conferred by the New South Wales Public Health Act 2010. Research ethics approval was not required.
Acknowledgements
We thank Justin Ellem, Gordana Nedeljkovic, Neisha Jeoffreys and Ian Carter from CIDMLS for technical advice. Referring laboratories and their staff, in particular Prof. Alison Kesson from The Children’s Hospital at Westmead, kindly submitted samples and provided measles-specific serology data. We thank staff from the Victorian Infectious Diseases Reference Laboratory (VIDRL) for providing measles virus genotyping data. VIDRL is the regional reference laboratory for the WHO Measles and Rubella
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2018, PathologyCitation Excerpt :Research ethics approval was not required as investigations of suspected or confirmed cases of measles was conducted under the legal authority conferred by the New South Wales Public Health Act 2010. The primers and probes used to detect all MeV variants were as previously published,3 whilst those used specifically to detect MeVA were adapted from Roy et al.8 Briefly, MeVA primers and probes were designed following the analysis of 31 sequences available on GenBank from Edmondston- and non-Edmonston-derived vaccine strains. Similar to Roy et al., our assay also uses a locked nucleic acid (LNA) probe, which targets a 23-base sequence similar to MeVA strains, but with four nucleotide differences from sequences of wild-type strains within the highly conserved amino terminus of the N gene.
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