Elsevier

Journal of Clinical Virology

Volume 70, September 2015, Pages 46-52
Journal of Clinical Virology

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

https://doi.org/10.1016/j.jcv.2015.07.004Get rights and content

Highlights

Abstract

Background

Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia’s largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination.

Objectives

To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler® platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples.

Study design

One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler® and LightCycler® RT-PCR platforms. The LightCycler® RT-PCR was used as the reference assay against which the SmartCycler® RT-PCR and IFA were compared.

Results

Using the LightCycler® RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler® and the LightCycler®-based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler® RT-PCR was 34.3% and 96.7%, respectively.

Conclusion

The performance of the SmartCycler® is comparable to the LightCycler® for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available.

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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