A valid assay requires that all positive and negative controls yield the expected results. The assay is not valid if any positive control is negative or any of the negative controls are positive. If any negative control has a positive result, the following steps should be taken:
- Immediately discard all working stocks of reagent dilutions and remake from fresh stock
- Bench surfaces, pipettes, and other equipment should be cleaned with bleach or a commercial product to destroy nucleic acids in the reagent setup and template addition work areas.
In real-time RT-PCR assays, a clinical sample that is positive by both virus- and cellular- gene specific RT-PCR (or RT-qPCR) is considered positive. In multiplex assays, if the virus-specific RT-PCR is positive, it is acceptable for the cellular gene to have a negative result- this does not invalidate the positive virus-specific result when expected results are obtained for the positive and negative controls.
Specimens that are negative by the virus-specific RT-PCR but positive by the cellular-gene specific RT-PCR are considered negative. If both the cellular-gene specific RT-PCR and the virus-specific RT-PCR have negative results, the quality of the RNA in the sample has may have been compromised and the result is reported as undetermined (inconclusive).
It is critical to carefully evaluate the data for each sample. The amplification curves must cross the set threshold for the virus-specific RT-PCR to be considered positive and visual inspection of the amplification curves is suggested to verify positive results. (See Figure 6.1).
Extremely weak positives (high Ct values close to the cut-off Ct) should be interpreted with caution. Ideally, the test samples should be tested in triplicate. If there are inconsistent results (positive/negative, positive/undetermined), the samples should be retested.
For conventional RT-PCR, the PCR products are usually visualized by agarose gel electrophoresis on a 1.0-2.0% agarose gel with an appropriate molecular weight marker. Figure 6.2, Rubella diagnostic RT-PCR control and wild-type products, shows the difference in migration of the amplicons from the rubella synthetic positive control and that from a wild-type rubella virus sample.
The instructions for agarose gel preparation and clean-up of PCR products are provided in Annex 6.6. Gels are stained by ethidium bromide or GelRedTM and bands are visualized on a uv transilluminator. Positivity is determined by the presence of a fluorescent band on the gel of the expected size. The image of the gel should be recorded with the results. If any of the controls fail to produce the expected result, the assay is invalid and must be repeated.
A positive result indicates that measles or rubella RNA was present in the clinical sample. However, if the suspected case had been vaccinated within 7-14 days prior to rash onset, the positive result may be due to a vaccine-associated rash. A set of criteria is available for identifying vaccine-associated rash cases (Chapter 4. Antibody detection methods for laboratory confirmation of measles, rubella, and CRS, text box section 4.3), when there was no potential exposure to wild-type measles or rubella virus.
In those instances where the patient received an MR/MMR vaccination following potential exposure to a confirmed case or was vaccinated as part an outbreak response, it is necessary to identify a vaccine strain (genotype A) by sequence analysis or by use of a validated vaccine-specific RT-PCR assay to discard the case. For more information on distinguishing vaccine strains from wild-type infection by sequence analysis, refer to Chapter 7. Molecular epidemiology of measles and rubella.
The interpretation of real-time RT-PCR test results must consider the possibility that the test result may not reflect the true case status of the suspected case. Not only can false positive (from a non-case) results arise due to cross contamination, a negative result may be obtained from a true case of measles or rubella. The abundance of virus-specific RNA is generally much lower in infected cells compared to the cellular RNA of the reference gene. Therefore, a positive signal from the reference gene may be obtained from specimens collected from true cases while the virus-specific real-time RT-PCR result is negative.
A ‘false negative’ result may be obtained due to:
- Poor sample collection, transportation or processing
- Partial degradation of RNA from excessive freeze-thaw cycles, reducing viral RNA below detection levels
- Timing of specimen collection
- Failure to follow the assay protocol or equipment failure
- Failure to use specified extraction kit and platform
Therefore, a negative RT-PCR result should be interpreted carefully. A negative RT-PCR result can be helpful to corroborate other negative laboratory results but should never be the sole basis for ruling out a suspected case. In addition, a negative RT-PCR result obtained from a suspected case with a doubtful (positive) IgM result does not provide support for concluding that a positive result for IgM was due to a nonspecific reaction. The final classification of suspected cases that are negative by RT-PCR with inconclusive IgM results (i.e. negative IgM from serum collected ≤3 days after rash for measles or ≤5 days after rash for rubella) must be based on clinical assessment or discarded based on laboratory confirmation of other rash-causing illness. In elimination settings, additional testing may be necessary or additional sample collection may be indicated. The recommendations for testing in elimination settings are provided in Chapter 8. Laboratory testing in support of measles and rubella surveillance in elimination settings.