Table 3 Potential issues when implementing a RITA, with recommendations and additional considerations

Recency assay

Assay availability may be limited

Discuss with assay manufacturer well in advance of launch of study the number of assays required.

This will require an understanding of the population to be studied and the anticipated confirmation rate.

Assay kit may not be able to be imported into country

Ensure import permits are in place at an early stage.

Delays to imports may affect performance of assay if it is not stored in appropriate condition when awaiting customs clearance.

Assay transport within country

Ensure cold chain is maintained for the assay when it is transported to local labs.

Minimising number of laboratories performing the assay may make this easier.

Sample types

Specimen transport within country

Specimens should be transported under cold chain. Serum/plasma specimens for LAg testing should be separated locally before transport. DBS specimens should ensure that the specimen is fully dried before transport and maintained with a desiccant.

Depending upon other tests required, different specimens may need to be transported under different conditions.

Training and performance

Before testing is undertaken staff should be trained in the performance of the assay

Although a standard EIA this assay has multiple pass/fail criteria and troubleshooting errors can be complex. All users should receive training from an experienced user before undertaking ‘real-world’ testing. CDC offer training panels to help users achieve competency in the assay and help should be sought from CDC.

This training will not only help support the testing procedure but also the analysis tools of the associated software.

Individuals should be monitored regularly to confirm compliance with the testing protocol. Testing laboratories must ensure that Standard operating procedures are in place for the assay detailing all steps and conditions undertaken in their lab to allow troubleshooting of issues and enhance data analysis.

Assay only requires relatively basic laboratory equipment however it is very sensitive to issues such as inadequate washing.

Ensure all required equipment is available and is fully serviced, maintained and calibrated. Equipment to be used should be itemised before testing begins and reviewed by an experienced individual to ensure it is suitable for use.

Service contracts may not be in place for some pieces of equipment so monitoring the performance of equipment is critical to ensure it is performing as expected. When considering equipment even ‘common’ items such as pipettes should be serviced and calibrated before use.

Additional items such as bloodletting equipment and Dried Blood spot cards are required

These are often outside of the control of the laboratory, but it should be ensured that the equipment used is of the right type, within expiry date and stored appropriately.

Details of all ancillary equipment should be recorded and available for inspection.

Reagents should be stored as per assays instructions for use (IFU).

Reagents are stored at a variety of temperatures and these must be adhered to. Temperature monitoring should be in place for all freezers and refrigerators.

Ensure that supplies such as water are appropriate for use following assay’s ‘IFU’.

In use reagents

Wash reagents must be made as per the assays ‘IFU’. Unless the whole kit is being used at one time then unused reagents must be returned to appropriate storage conditions as soon as possible.

Testing must be performed in accordance with the assays ‘instruction for use’

LAg assay is a generic term and at least two manufacturers supply a version of the assay. It is critical that the correct method is used for each assay.

As well as two manufacturers there are different versions of the kit dependent on sample type being used plasma/serum or DBS. The appropriate method must be followed for these.

Quality control

Temperature control

LAg assays are very prescriptive on incubation times and temperatures. Assays should be performed using incubators with precise and recorded temperatures rather than incubated in the open lab regardless of the current temperature of the lab.

Regular monitoring of equipment temperatures should be recorded and incubators confirmed to have reached temperature before starting assay.

Internal quality control

Although the assays come with control material, users should follow good laboratory practice and include some specimens of known reactivity in every test to ensure reproducibility over time.

These results should be plotted and analysed to look for any trends in performance.

Analysis of quality control data

The manufacturer supplied analysis software will provide quality control data on the assay performance. Users should ensure that they enter data correctly and ensure upper reading OD of the spectrophotometer is accurately entered. Users should review data on their own internal quality control independently as this is not captured longitudinally by company software.

External Quality Assurance

Each testing laboratory should partake in an external quality assurance programme for the assay. This will help provide confidence in the results issued by each laboratory. The programme for this assay is available via EQAPOL.

Assay failures

Laboratories should record and share any failed runs with assay specialists. This will help to identify if there is any systematic error occurring with the assay and enhance the ability to troubleshoot the assay.

Users should ensure that details of all equipment used (Including pipettes etc) is recorded and the items are identifiable by serial number. Batch number of assay should be recorded (this will be performed by the company supplied software if used).

Assay linked analysis software

Users should ensure they are using the correct analysis software associated with the assays. Each assay has different validation criteria and use of the incorrect manufacturer’s software may lead to errors in assay interpretation and validation.

Users should also be aware to use the correct software for plasma/serum specimens and for DBS specimens as these differ.

Confirmatory testing

Confirmatory testing must be performed from a freshly diluted specimen. It must not be performed from the dilution prepared for the screening assay.

Reviewing confirmatory test data can help determine how well an assay is being performed as the replicates should be very close. A wide range of values on the confirmatory test may indicate poor pipetting competency of the tester or poor reliability of the pipettes.

Unusual results

All unusual results should be investigated, and retesting undertaken if warranted. Unusual results may include specimens which offer a low OD (which require retesting to confirm the specimen contains antibodies to HIV-1) or samples where the screening ODn is significantly different from that of the confirmatory test. Investigations should be undertaken to confirm that the correct specimen was retested (or tested initially).

Many assays will not differentiate HIV-1 from HIV-2 so care should be taken where HIV-2 is prevalent.

Reporting results

Confounding factors

Users should be aware that a number of factors can affect the performance of the test and these should be considered when analysing data. These include factors such as anti-retroviral treatment of individuals and HIV subtype prevalence.

Assay specific performance data

Users should be very clear as to which assay was used and the cut-offs applied to their data as this impacts on the period in which recent HIV infection can be inferred by the assay. This is also important where multiple HIV-1 clades are prevalent.

Results should not be issued without being part of an algorithm to reduce the potential for misclassification.

Over-interpretation of the data

Users should be aware of the limitations of the assay and not over interpret the data. For example, a very low ODn may indicate a very recent HIV infection or that the individual is not HIV infected. As per the algorithm these specimens should be submitted for HIV diagnostic confirmatory testing.

Application of data trends should take into account potential confounders such as changes in the population, the application of different HIC screening tests over time and the use of different LAg assays in previous surveys.