10.2 Four quality indicators with direct support and input of laboratory

Mick Mulders

Four of the eight indicators (highlighted above) are directly related to the management and performance of the laboratory but also require sufficient resources and performance of a sensitive system for detection and investigation of suspected cases of measles and rubella. Each of these four indicators is discussed in greater detail in the sections that follow.

10.2.1 Reporting rate of discarded non-measles non-rubella cases

While this indicator measures the adequacy of surveillance for rash illnesses that could include suspected cases of measles and rubella, the tests performed by the laboratory are critical for appropriate classification of febrile rash illnesses as non-measles and non-rubella cases. The midterm report for the Strategic Plan 2012-2020 recommended that specimens that test IgM negative from suspected measles cases be tested for rubella IgM, if both are not tested for at the same time [3]. While evaluation of IgM in serum has been the gold standard for classification of suspected cases, as discussed in Chapter 8, IgM testing has limitations in elimination settings. As disease becomes rare, case classification will increasingly rely on molecular testing to support IgM results and genetic analysis to accurately identify vaccine-associated rashes (see chapter 7).

10.2.2 Laboratory confirmation

Calculation of the laboratory confirmation of measles and rubella infection applies primarily to testing for virus-specific IgM, usually by EIA. However, the use of RT-PCR for case confirmation, (Chapter 6), has become a routine method in many countries as an adjunct method for case confirmation. Therefore, the laboratory confirmation indicator may involve calculation of the proportion of suspected cases that were tested by each method of detection at the national level. Ideally, the proportion of suspected cases that were tested by each method should be calculated separately. Collection of an adequate specimen, therefore, may include collection of specimens for viral detection, particularly for RT-PCR.

Adequate samples for antibody detection must be collected within 28 days after onset of rash. The guidelines for providing optimal specimens for testing are described below:

  • A blood sample collected by venipuncture in a sterile tube with a volume of 5 ml for older children and adults. For infants and younger children, attempt to collect 1 ml of blood (may be collected by foot or finger stick) The stated volumes above are the goal; specimens should not be considered inadequate if volumes are sufficient for testing as determined by the laboratory
  • A dried blood sample, at least 3 fully filled circles (4 filled circles are ideal) on an appropriate filter-paper collection device
  • An oral fluid sample using a sponge collection device that is rubbed along the gums for >2 minutes to ensure the device is thoroughly wet with a total IgG level of greater than 3 ug/ml
Laboratory confirmation indicator:

The proportion of suspected cases (see caveat below regarding the denominator) with adequate specimens for detecting acute measles or rubella infection collected and tested in a proficient laboratory. As indicated above, the numerator consists of cases that are laboratory confirmed by specific antibody detection, but molecular testing may be an additional method of confirmation. The indicator should reflect testing of suspected cases performed by a proficient laboratory that has either been accredited by WHO and/or has an established quality assurance programme with oversight by a WHO accredited laboratory.

The target is to provide laboratory confirmation for ≥80% of suspected cases. The calculation of the proportion used to evaluate the indicator of laboratory should exclude the following two groups from the denominator of suspected cases.
  • Suspected cases of measles or rubella that are not tested by a laboratory and are confirmed as measles or rubella by epidemiological linkage
  • Suspected cases that are discarded* as non-measles and non-rubella by epidemiological linkage to another laboratory-confirmed communicable disease case

*Additional criteria may be used for discarding cases, such as demonstration of a vaccine strain from recently vaccinated cases (see criteria for a vaccine-associated rash, chapter 4)

10.2.3 Viral detection

The inclusion of an indicator for viral detection reflects the critical contribution of the molecular characterization of measles and rubella viruses. While viral detection can provide confirmation of suspected cases as discussed in the previous section for the indicator of laboratory confirmation, this indicator is designed to capture two activities that promote molecular surveillance:

  1. The proportion of identified chains of transmission for which adequate samples for viral detection are collected (reflecting the timeliness and consistency of collection). The numerator is the number of chains of transmission for which adequate samples have been submitted for viral detection. The denominator is the number of chains of transmission identified.
  2. The percentage of all chains of transmission, identified during a calendar year, that have been successfully characterized by genetic analysis (identification of genotype/named strain). The denominator is the same as in 1) above, while the numerator is the number of chains of transmission for which a genotype has been identified.

Therefore, this indicator can be used to assess both process (adequate sample collection) and outcome (completeness of molecular surveillance for genotypes associated with outbreaks or chains of transmission that are identified in the country). This latter laboratory component of the indicator is designed to demonstrate that molecular data is sufficient to demonstrate interruption of transmission of endemic viruses, one of the lines of evidence for verification of elimination. If elimination has been achieved, genetic analysis of viruses from sporadic cases and small outbreaks provides evidence for maintenance of elimination. The timing for an adequate sample varies according to the type of specimen and the method of detection:

  • For virus isolation, throat or urine samples are collected within 5 days after rash onset
  • For virus detection using molecular techniques: adequate throat samples are those collected up to 14 days after onset of rash; oral fluid samples may potentially yield a positive signal by RT-PCR up to 21 days after onset of rash

An adequate sample, as outlined above, may not contain sufficient virus-specific RNA or viable virus. Specimens collected closer to rash onset will provide higher success rates for both purposes. However, the conditions under which the specimen was collected, transported or processed may result in a loss of virus titre and degradation of RNA. In addition, specimens that meet the requirements stipulated above for an adequate sample have an insufficient amount of intact RNA for sequencing even though a positive signal was obtained by real-time RT-PCR. However, for assessing the process component of this indicator, a sample is considered adequate if the timing of collection is within the acceptable range for the specimen type. For more information on sample collection, refer to chapter 3.

The goal that has been established for the indicator for virologic surveillance is intended to demonstrate that all chains of transmission recorded in one calendar year received 1) the timely and appropriate response to collect adequate samples for viral detection and 2), the successful identification of the genotype associated with the chain of transmission (or named strain if applicable, per regional surveillance guidelines*). If a laboratory performs virus isolation only, the infected cell lysate or RNA extract should be promptly submitted to an appropriate laboratory for genetic analysis.

The collection of 5-10 virologic samples early in the outbreak is recommended to ensure that the genotype can be identified. Additional samples should be collected on a monthly basis should cases continue. Frequent communication regarding the epidemiologic characteristics of the outbreak between field epidemiologists or programme staff and the laboratory can help determine whether additional specimens should be collected to provide adequate genetic characterization of the outbreak. In elimination and near-elimination settings, the ability to track transmission pathways by phylogenetic analysis may be greatly enhanced by sequence analysis of multiple cases during an outbreak with the aid of extended sequencing methodologies (see chapter 7). If the outbreak expands into other areas, or the epidemiologic links are not well-defined, additional samples should be collected from the new cases.

*Refer to regional surveillance guidelines. For example, in the European Region, identification of a genotype may not supply adequate discrimination between sequences for tracking transmission pathways. The template for country reports includes a field to be completed with the named strain within the genotype (if available) as a part of the genetic data [4].

A chain of transmission describes a focalized outbreak that is undergoing an active investigation of contacts and identification of additional cases that are traceable to earlier confirmed cases. The definition of an outbreak for disease activity classification depends on national surveillance guidelines but usually consists of a minimum of either two or three epidemiologically-linked cases. However, it is understood that a single confirmed case must receive a vigorous investigation as a potential outbreak. This is especially true of rubella, since up to 50% of cases may be subclinical or inapparent.

If the epidemiologic evidence supports a single chain of transmission, the identification of a genotype from any case in the chain/outbreak is sufficient for assignment of a genotype for the chain of transmission. It should be noted that the source or origin of infection is inherently shared among cases identified within a single chain of transmission. However, successful designation of a genotype associated with a chain of transmission (indicator for viral detection successfully met) does not directly coincide with a known source for the outbreak or chain of transmission. The interpretation of data on MeaNS and determination of source of infection is discussed in chapter 7.

The rate of viral detection is a key surveillance indicator that is assessed in the verification process. The performance of this indicator depends on the timely exchange of information between the epidemiologic team and the scientists that manage the work flow, prioritize sequencing activities, and have responsibility for reporting genotypes. The epidemiologic data necessary to determine which specimens correspond to different chains of transmission can help guide decisions on testing and indicate whether adequate specimens have been submitted for characterization of genotypes from each chain of transmission.

Figure 10 illustrates how a timely epidemiologic investigation can facilitate the efficient use of time and resources. The laboratory can identify which samples should be prioritized for sequencing to ensure that all chains of transmission can be genotyped or, if necessary, request additional samples. In the lower panel (B), the epidemiological investigation is not conducted in a timely manner or there is a delay in the arrival of the information to those in the laboratory. The genotyping efforts are conducted randomly on specimens received without the benefit of knowing how many chains of transmission there are, or which cases and specimens are linked. A retrospective determination of the genotyped cases reveals that only three of the five chains were successfully characterized by genotyping.

10.2.4 Timeliness of reporting laboratory results

Countries nearing elimination of measles and/or rubella should investigate all suspected cases and obtain a clinical specimen for laboratory testing. Once the case investigation form has been completed and laboratory test results are available, suspected cases should be classified (or additional testing performed) according to the flowchart in chapter 8 (Figure 8 http://www.who.int/immunization/monitoring_surveillance/burden/laboratory/figure_8.pdf">).

Timeliness of reporting indicator:
This indicator is comprised of two parts:

  1. Proportion of surveillance units reporting to the national level on time, with a target of ≥80%.
  2. Proportion of countries reporting to their WHO Regional Office on time, with a target of 100%.

Importantly, “reporting” includes the requirement that discarded cases (non-measles, non-rubella) are reported to the national level with a target rate of ≥2 cases per 100,000 population per year. To meet the requirement for on time reporting, the information must be received by the date requested or within the timeframe established for the report. For more information regarding the necessary information to accompany the report, see chapter 11.