Specimens for serologic testing (serum or oral fluid) and clinical specimens for RT-PCR (oral fluid, nasopharyngeal (throat), urine) should be collected at the first contact with a suspected case of measles or rubella. Recommendations for collection of specimens for antibody testing and for molecular testing to provide high-quality surveillance for measles and rubella are presented in chapter 3. The collection of both types of specimens is recommended for accurate case classification. The flowchart for testing in elimination settings (Figure 8) provides options for serologic and molecular testing that may be appropriate for laboratory confirmation of suspected cases of measles and rubella. Figure 8A consists of the steps involved in RT-PCR testing. In Figures 8B and 8C, routine IgM testing as well as additional options for serologic testing are included if RT-PCR results are inconclusive.
Because not all SNLs or NLs have the capacity to perform all types of tests, especially those requiring specialized reagents, protocols should be in place for referral of samples when additional testing is deemed appropriate. The flowcharts in Figure 8 are intended to provide a comprehensive guide, however each country will need to tailor the approach to resolve challenging cases depending on the particular setting in which the suspected case occurs, the available resources, and the epidemiologic information. The appropriate NL or RRL should be consulted prior to referral of samples that have been identified for additional testing. Examples of additional and/or specialised tests that may aid in the final classification of suspected cases include:
- Kits or reagents for the detection of IgM or virus-specific RNA for alternative causes of rash illness
- Virus-specific IgG EIA kits
- Quantitative/semi-quantitative IgG assays are required for evaluation of a diagnostic rise in titre between paired serum specimens
- Rubella IgG avidity assays
- Measles IgG avidity assays
- Measurement of virus-specific neutralising antibody
- Enhanced sensitivity for detection of virus-specific RNA using real-time RT-PCR
- Detection of vaccine viruses using a real-time RT-PCR protocol that targets vaccine-specific nucleotide sequences
- Sequence analysis to rule out wild-type virus (confirm suspected vaccine-associated rash)
- Kits or reagents for the detection of IgM or virus-specific RNA for alternative causes of rash illness
- Virus-specific IgG EIA kits
- Quantitative/semi-quantitative IgG assays are required for evaluation of a diagnostic rise in titre between paired serum specimens
- Rubella IgG avidity assays
- Measles IgG avidity assays
- Measurement of virus-specific neutralising antibody
- Enhanced sensitivity for detection of virus-specific RNA using real-time RT-PCR
- Detection of vaccine viruses using a real-time RT-PCR protocol that targets vaccine-specific nucleotide sequences
- Sequence analysis to rule out wild-type virus (confirm suspected vaccine-associated rash)
Many of the laboratories in the GMRLN have established protocols for differential testing to rule out alternative causes of rash illness that are typical in the country or region (e.g., dengue, chikungunya, zika). The decision to proceed with additional testing, particularly testing that would require referral to a RL or GSL, should be made in consultation with local epidemiologists and the RLC.
8.4.1 Guidelines for testing paired serum specimens for IgG titre measurements
For primary cases of measles or rubella, the follow-up serum specimen should be collected 10-21 days following collection of the initial (acute) serum specimen (which should be collected within 7 days after onset of rash). The serum specimens must be tested in parallel (i.e., tested together, in the same assay). The optical density values measured by the IgG EIA must fall into the acceptable range according to the kit parameters. Demonstration of a diagnostically significant rise (a 4-fold rise or as stipulated by the algorithm specific for the kit that converts OD values to titres) between paired specimens confirms the case.
In some instances, there may be an observed rise in titre but the increase in titre does not reach the threshold required by the kit parameters (or 4-fold) for confirmation of infection. The ability to demonstrate a significant rise in titre is dependent on the appropriate interval between collection of the serum specimens. Because of individual differences in immune responses, the ideal interval may vary to some extent. The uncertainty and logistical difficulty associated with collection of paired serum specimens requires a careful evaluation to justify ruling out a suspected case based on the absence of a significant rise in titre.
A rise in IgG antibody titre among individuals with a measles reinfection case is rapid and the results obtained from testing paired specimens can vary. Testing paired serum specimens with a gap of 2-3 weeks between blood collections may yield a diagnostically significant rise in titre. However, it is also possible that no rise is apparent, or a slight decrease in titre may be observed. This is due to the fact that the levels of antibody may peak much closer to rash onset (e.g., 3-10 days post rash onset) among measles reinfection cases [9].
If a reinfection is suspected, paired serum specimens can both be collected during the acute phase, with an interval of 3-5 days between collection of the specimens. Measles reinfection cases typically produce levels of IgG antibody that are much higher than what is generated following a primary response to measles. The conversion from the OD values obtained by EIA to titres is only valid within a specified range of OD values (per the specific manufacturer kit insert), which were developed and validated for the IgG assay using serum panels with much lower reactivity.
8.4.2 Criteria for discarding a suspected case of measles or rubella
To discard a case with a possible false positive IgM or equivocal result, one of the following test results or circumstances must be documented:
- Repeat testing for IgM on the same specimen using a validated method is IgM negative
- The first serologic specimen is negative for virus-specific IgG and a second specimen collected at least 10 days after rash onset, is also IgG negative when tested in the same assay performed together on the same day
- Repeat testing for IgM on the same specimen remains equivocal but virus-specific IgG is positive. This applies to rubella suspect cases as well as measles suspected cases unless a measles reinfection is suspected*
- The patient was vaccinated 7-14 days before rash onset (unless it was for post-exposure prophylaxis or outbreak control). See additional criteria for a vaccine-associated case provided in chapter 4
- Appropriately-timed, paired serum specimens do not show any change in IgG titre**
- The cause of rash was demonstrated to be due to an alternative etiologic agent by additional testing*** (e.g., chikungunya, dengue or parvovirus B19)
- Repeat testing for IgM on the same specimen using a validated method is IgM negative
- The first serologic specimen is negative for virus-specific IgG and a second specimen collected at least 10 days after rash onset, is also IgG negative when tested in the same assay performed together on the same day
- Repeat testing for IgM on the same specimen remains equivocal but virus-specific IgG is positive. This applies to rubella suspect cases as well as measles suspected cases unless a measles reinfection is suspected*
- The patient was vaccinated 7-14 days before rash onset (unless it was for post-exposure prophylaxis or outbreak control). See additional criteria for a vaccine-associated case provided in chapter 4
- Appropriately-timed, paired serum specimens do not show any change in IgG titre**
- The cause of rash was demonstrated to be due to an alternative etiologic agent by additional testing*** (e.g., chikungunya, dengue or parvovirus B19)
* Often, it is the epidemiological circumstances which lead to suspicion of measles, rather than the clinical presentation, since symptoms exhibited by an individual with a measles reinfection may be less severe, and the rash may not follow the usual progression.
** The rule-out of a suspected case based on the absence of a rise in IgG should be carefully evaluated in conjunction with the epidemiologic setting and the clinical presentation.
*** Either RT-PCR or demonstration of a seroconversion is required to confirm a dengue infection. An IgM positive result for dengue may be obtained months after the acute dengue infection.