Guidance

Infectious diseases in pregnancy screening programme laboratory handbook

Updated 25 May 2022

Applies to England

It is important to note that the terms ‘woman’ or ‘mother’ as used in this document encompass all gender identities and are intended for anyone who is pregnant.

1. Introduction

This handbook is for laboratories that provide services for the NHS infectious diseases in pregnancy screening (IDPS) programme. This includes both screening laboratories and those providing confirmatory testing. This laboratory handbook includes reference to the national guidelines, standards and pathway screening requirements specification for the NHS IDPS programme that relate to antenatal screening for human immunodeficiency virus (HIV), hepatitis B virus (HBV) and syphilis. This handbook was developed by the IDPS laboratory advisory group.

Each national NHS screening programme has a defined set of standards that providers must meet to ensure local services are safe and effective.

Screening standards provide:

• reliable and timely information about the quality of the screening programme
• data at local, regional and national level
• quality measures across the screening pathway without gaps or duplications

They also ensure a consistent approach across screening programmes and that data collection is beneficial.

Quality assurance (QA) is the process of checking that these standards are met and encouraging continuous improvement.

The IDPS screening laboratories must:

• be accredited by the UK Accreditation Service (UKAS) to ISO 15189:2012 medical laboratories – requirements for quality and competence
• meet the IDPS laboratory QA evidence requirements mapped to ISO 15189:2012
• participate in external quality assurance (EQA) schemes accredited to ISO 17043:2010 conformity assessment – general requirements for proficiency testing
• use reference laboratories accredited to ISO 15189:2012 for confirmation of screen positive results when this is not available in-house

UKAS will look at both ISO 15189 and the screening requirements on behalf of the screening quality assurance service (SQAS).

1.1 Related documents

This handbook should be read in conjunction with:

• the IDPS programme standards
• the IDPS pathway requirements specification
• the IDPS programme handbook
• the operating model for quality assurance
• Managing safety incidents in NHS screening programmes
• NHS population screening explained
• the principles of good laboratory practice

2. Infectious diseases screened for

The UK National Screening Committee (UK NSC) recommends systematic population screening in pregnancy to all eligible women for:

• HIV
• hepatitis B
• syphilis

The objectives of the IDPS programme are to:

• reduce the risk of vertical (mother to child) transmission of HIV, hepatitis B and syphilis
• ensure that women with HIV, hepatitis B and syphilis are identified early in pregnancy to facilitate appropriate timely assessment and management of their health
• facilitate an informed personal choice
• facilitate appropriate and timely neonatal referral and management

The UK NSC periodically reviews the 3 infections. Screening for HIV, hepatitis B and syphilis are all recommended as they meet internationally recognised criteria and a rigorous evidence review process. They are identified as viable, effective and appropriate for screening as there are effective interventions that can be made in pregnancy, at delivery and postnatally to significantly reduce the risk of transmission. Prevalence in England is low but screening is regarded as cost-effective because of the lifetime costs of these infections.

Depending on the local population, some smaller antenatal services and laboratories may rarely encounter women with screen positive results. It is important that relevant protocols are in place and can be readily accessed as staff may not be familiar with what is required.

All of these infections can be acquired by the mother during her pregnancy. A negative antenatal screen is ‘negative now’ (at the time the blood sample was taken) and retesting may be indicated if risk factors are identified or clinical features seen in the mother or infant.

2.1 HIV

HIV is transmitted through virus-containing bodily fluids and can be transmitted from a woman living with HIV to her infant during pregnancy, delivery and whilst breastfeeding (vertical transmission). The level of risk is dependent on the amount of virus in the blood, genital tract or breast milk. The risk of transmission is highest at delivery, although in utero transmission can occur with high viral load, for example during HIV seroconversion, and if there is a concomitant sexually transmitted infection such as syphilis. The risk from breast feeding continues until the infant is weaned off the breast. Historical data shows that without any interventions the chance of vertical transmission among diagnosed women in the UK was 25.6%. With effective interventions the risk of transmission is much reduced, and in the UK transmission is now less than 0.3% overall and in women with an undetectable viral load it is 0.14%.

The risk of vertical transmission is minimised by women receiving effective antiretroviral treatment in pregnancy (viral load undetectable) and post-exposure prophylaxis (PEP) of the infant. The duration and specific drugs used for PEP are dependent on the maternal viral load and the HIV resistance profile. If the maternal viral load is undetectable, then additional interventions such as elective caesarean section will be unnecessary. Breastfeeding is not recommended but will be supported in women on effective treatment with additional monitoring of both mother and baby.

Coverage of HIV screening among women who attend for antenatal care is more than 99%. New HIV diagnoses have declined significantly in the last few years and in antenatal clinics are low at 0.013%. Approximately 90% of pregnant women with HIV are now aware of their HIV status, with more than 80% of those on treatment before pregnancy. However, there are still women whose diagnosis is first made by antenatal screening and offering and recommending HIV testing to all pregnant women remains relevant. In addition, some women may not be accessing HIV care currently and will need re-engagement in HIV care.

2.2 Hepatitis B

Hepatitis B virus (HBV) is highly infectious and is present in all bodily fluids. Vertical transmission is largely attributed to exposure during or after delivery and trans-placental infection during pregnancy is thought to be rare. The risk of vertical transmission of hepatitis B is variable and dependent on the phase of the maternal infection and the woman’s level of infectivity. In the absence of intervention, vertical transmission occurs in 70% to 90% of pregnancies where the woman’s hepatitis B e antigen status is positive and in about 10% where the woman’s e antigen status is negative. This increased risk of transmission is related to the HBV viral load which is usually high where the e antigen status is positive and lower where the e antigen status is negative. However, there are exceptions and some women where the e antigen status is negative will have high viral load and are at increased risk of vertical transmission.

Compared to hepatitis B acquisition in adulthood, infants have a much higher risk of failing to clear the virus and most (more than 90%) infants become chronic carriers and have a high lifetime risk of death from cirrhosis or liver cancer.

Vertical transmission of hepatitis B is preventable in 90 to 95% of potential cases through a combination of vaccinating the infant starting at birth (for all babies born to women with hepatitis B) and hepatitis B specific immunoglobulin (for infants at high risk of infection which is determined by maternal serological infectivity markers, hepatitis B DNA/viral load and the baby’s birth weight). Increasingly women with high hepatitis B viral load will be offered antiviral drugs in the third trimester of pregnancy to reduce their viral load and further reduce the risk of transmission. Further information on the vaccination programme can be found in the Green Book.

Overall prevalence of HBV in England is low but infection is unevenly distributed with some areas of the country having a higher prevalence of infection than others. Approximately 0.4% of pregnant women in England have hepatitis B. Coverage of antenatal screening remains high at over 99%.

2.3 Syphilis

There has been a substantial increase in the number of infectious syphilis diagnoses made in England and there is concern that this increase will be reflected in pregnant women, potentially resulting in an increase in congenital syphilis. Syphilis screening coverage in pregnancy is more than 99% at present and it is clearly important to maintain this high antenatal screening coverage. Although the UK incidence of congenital syphilis is below the World Health Organization (WHO) elimination threshold (less than or equal to 0.5/1000 live births), congenital syphilis continues to present a complex clinical, social and public health problem.

Most cases of syphilis transmission occur transplacentally, although transmission during delivery is also possible. Unlike HIV or hepatitis B the fetus can be both infected and affected during pregnancy resulting in late fetal loss, premature delivery, low birth weight and neonatal death. Signs and symptoms of congenital syphilis in the infant can include jaundice, severe anaemia and neurological complications. Maternal treatment and the immunological response of the fetus can affect the clinical presentation in the baby. There is wide spectrum of disease and some infants have no obvious signs or symptoms and are not investigated and diagnosed if maternal syphilis infection is not identified.

Vertical transmission of syphilis can be significantly reduced if women receive adequate and prompt antibiotic treatment during pregnancy. Although vertical transmission can occur at any stage of syphilis and any stage of pregnancy, the risk is highest in early maternal infection and when delivery of the baby is within 30 days of maternal treatment. Nearly all pregnant women with untreated primary or secondary syphilis experience adverse outcomes, such as neonatal deaths and stillbirths, or congenital syphilis in surviving infants. Recent reviews of cases of vertical transmission of syphilis have shown that some women acquire syphilis and develop primary infection after an initial negative screening result. This highlights the importance of offering repeat testing to women who were at risk of exposure to syphilis and/or who have relevant symptoms, at any stage of pregnancy.

3. Personal informed choice

All screening is an individual choice. Everyone must be given the opportunity to make a personal informed choice about whether or not to be screened. The decision must be based on an understanding of:

• why they are being offered screening
• what happens during the test
• the benefits and risks of screening
• the risks of not screening
• the potential outcomes (including types of result, further tests and treatment)
• what happens to their screening records

If a woman is provided with the above information about the programme and chooses not to have screening for any or all of the 3 infections, then this is a valid choice and must be respected. A formal re-offer of screening must be made by the screening team by 20 weeks gestation which ensures that the woman has received appropriate information when making the decision to decline screening. Women who book after 20 weeks gestation and decline screening must be re-offered within 2 weeks.

4. Organisation of screening services

Providers must have a risk assessment of the entire pathway and protocols defining responsibility for all aspects of the process. Laboratories must have protocols detailing receipt of samples, analytical procedures and reporting of results in a timely manner. If some or all samples are referred to or received from other laboratories, there must be agreements covering all of these aspects. Additionally, there must be an agreement outlining who is responsible for the provision of screening data, standards and key performance indicators (KPIs).

Where samples are sent to other laboratories for confirmatory testing the laboratory must make sure they meet and comply with screening programme requirements.

All results should be reported to the maternity services by or before 8 working days of sample receipt. To achieve this a rapid service for confirmatory testing must be provided to avoid delays in reporting. The turnaround time is the same irrespective of whether confirmatory testing is performed in-house or sent to a reference laboratory.

5. Multidisciplinary working

The screening and management of infections in pregnancy involves a number of specialities and so there is a need for effective multidisciplinary working. This is supported in clinical guidance by the British HIV Association (BHIVA) and the British Association for Sexual Health and HIV (BASHH). A multidisciplinary team (MDT) approach enables clear and timely communication between professionals and robust working relationships to ensure high quality, women centred care can be delivered.

The laboratory team is integral to the delivery of the screening pathway and functions of the IDPS MDT. There must be representation from the laboratory team on trust screening governance groups.

6. Laboratory management requirements

The initial screening tests for all 3 infections (HIV, hepatitis B and syphilis) must be performed in a single laboratory or within a single multidisciplinary pathology department or network to make sure there is efficient coordination of processing the sample and reporting the results.

The laboratory must:

• have oversight and leadership of screening
• include a named senior biomedical scientist in the management structure for screening
• have a named, discipline-specific clinical lead from a virology, microbiology or infectious diseases background, who has passed the FRCPath Part 2 by examination (or has a recognised examination if overseas-qualified), who has day-to-day responsibility for the IDPS service.
• have the clinical head of pathology, clinical director or laboratory director as the named person who is ultimately accountable for the service
• have a viable contingency plan to continue the provision of IDPS in the event of any failures to the laboratory service (contingency plans must make sure specified test turnaround times are achieved)
• participate in the cross-organisational and multidisciplinary arrangements for the governance, management, communication and development of the screening pathway, including:
  • having clear communication arrangements with users and commissioners of the service, including public health
  • sharing information on laboratory screening performance, quality indicators and incidents
  • participation in screening safety incident investigations as appropriate

The laboratory management review must specifically include review of the laboratory’s performance in the delivery of the IDPS programme.

The laboratory leadership must ensure there are effective procedures in place to assess and manage safety and performance, including:

• documented risk management policy for the laboratory as part of the overall risk management arrangements for the screening pathway
• compliance with the NHS screening programmes guidance for managing safety incidents
• provision of KPI data and screening activity data to NHS screening programmes

7. Laboratory quality management system

The laboratory quality management system (QMS) must incorporate all the requirements of the IDPS laboratory service. The laboratory must have documented standard operating procedures for the following processes, agreed with relevant services, for how screening samples are monitored and managed. These must include identified responsibilities and failsafe arrangements.

7.1 Receiving and processing samples to enable matching against the cohort of women who have accepted screening

All women booking for antenatal care must be offered and recommended screening for HIV, hepatitis B and syphilis in every pregnancy regardless of previous results or treatment. Women have the choice to accept all screening tests, to decline all tests or accept the offer of just one or 2 tests.

The sample must be clearly identified as an antenatal screening sample to:

• enable screening laboratories to identify antenatal samples as distinct from other samples they receive
• match these samples to a specific maternity service

The laboratory must have clear acceptance and rejection criteria for samples to include:

• essential clinical and demographic information
• adequacy of sample labelling
• sample quality

The request form or electronic data request fields must be compliant with the minimum data fields (see ‘Minimum data field table’ under ‘Documents’ heading).

Samples received in the screening laboratory must be recorded as received on the laboratory information management system. Samples should be ‘logged’ as antenatal samples, assigned a laboratory accession number and booked for the appropriate tests according to the request within 24 hours of receipt in the laboratory.

It may be necessary to return to the original sample tube to check the identification details and for confirmatory testing. For this reason, it must be standard practice to make sure that any labels attached to the sample on receipt in the laboratory do not obscure the woman’s identity (either written or attached label).

If samples are passed between laboratories or sites within the department there must be a process to make sure samples are tracked and that all the requesting information is sent with the sample.

It is recommended that women already known to be living with HIV or hepatitis B are offered rescreening in any subsequent pregnancy (including confirmatory testing). This enables the current maternity service to have the screening results and also provides a failsafe for ensuring prompt referral to specialist services.

If a woman discloses that she is positive for HIV or hepatitis B but declines rescreening for that particular infection, screening for the other infections must still be offered and recommended. The information that she is positive for HIV or hepatitis B must be recorded on the laboratory request form by the requestor as a ‘known positive’.

7.2 Reporting positive or equivocal screening results

Positive or equivocal screening results must not be reported verbally (with the exception of urgent results as discussed below), in written or electronic form to the maternity service (for example screening team, specialist midwife or clinical nurse specialist) until confirmatory tests are completed on the screening sample.

Positive or equivocal screening results must only be reported on the screening sample for HIV, hepatitis B and syphilis following confirmation of the result using appropriate analytical methods.

7.3 Direct reporting of confirmed positive results

In addition to electronic reporting, laboratories must directly inform the screening team or infectious diseases midwife of all confirmed positive results, preferably by use of a generic email address or by telephone. This is required to minimise the delay between reporting and informing the woman of her results and referring to specialist services. The laboratory team must record when and to whom they gave the result.

In some laboratories confirmatory testing is sent to a reference laboratory. Where this occurs the screening laboratory must have a process in place to track samples from the time they are sent away to the receipt of results and to follow-up samples where the turnaround time from the reference laboratory is delayed.

Requesting a repeat sample ‘to confirm patient identity’ before informing the woman of her results and/or before referral to specialist services is not required. See ‘Repeat samples for confirmation’ heading in section 8.1.

7.4 Inconclusive results and repeat samples

There must be a process in place between the laboratory and the screening coordinator or infectious diseases midwife to alert them to an inconclusive result and the need for a repeat sample to exclude recent infection.

An inconclusive result is one where the screening result is not supported by the relevant confirmatory tests. When the results are inconclusive after confirmatory tests on the screening sample a repeat sample must be requested. The overall results are most likely to be inconclusive when the initial screening assay gives an equivocal or low positive result. The repeat sample must be taken a minimum of 2 weeks from the date of the initial sample. This is particularly relevant to HIV and syphilis; inconclusive results are less frequently encountered with HBV screening. Guidance on reporting initial and repeat samples is given for the individual infections in the technical requirements sections. Laboratories must aim to give a definitive result on the repeat sample.

Where a screening laboratory does not undertake in-house confirmation a sample testing equivocal, positive or detected in the screening assay will be sent to a reference laboratory for confirmation. Although the reference laboratory may report that the sample has tested negative or not detected in their assays, the screening laboratory should also take their own result into account when reporting. This combination of results would then be reported as inconclusive and a repeat sample must be requested.

Laboratories must directly inform the screening coordinator or screening team of inconclusive results on the initial screening sample that requires a repeat sample taken a minimum of 2 weeks later. They should use the same process as for confirmed screen positive results.

For most women, the repeat sample will exclude recent infection as the serological markers will not have changed. To avoid unnecessary distress and anxiety referral to sexual health services is not recommended unless the repeat sample confirms an infection. Result reporting must be clear so that the screening team can confidently follow this recommendation.

7.5 Follow-up list of relevant results

A follow-up list of relevant results must be produced each week (or more frequently by local agreement) and sent to the screening coordinator, infectious diseases midwife or other nominated person.

The follow-up list must include all positive and inconclusive results and all samples which were not processed because the request is incomplete or the sample inadequate or insufficient.

7.6 Urgent testing for unbooked women or women with no screening results presenting in labour

Tests must be performed urgently for all women presenting in labour who are either unbooked or with no evidence of screening results from a UKAS accredited laboratory.

The test turnaround time of by or before 8 working days does not apply for women without screening results who are:

• in labour or who have just delivered
• admitted to maternity services and have a high risk of premature labour

In order to expedite testing the maternity service must liaise directly with the laboratory, usually by telephone, to ensure the laboratory has the necessary clinical information to inform prompt analyses. Maternity services must not rely only on writing ‘urgent’ or similar on the request form or flagging ‘urgent’ on an electronic request.

The majority of these urgent screening results will be ‘not detected’ and can be reported promptly. If any of the screening tests are ‘detected’ there needs to be an urgent clinician to clinician conversation. This should be between the laboratory clinician and the on-call obstetrician and paediatrician. This is the only circumstance in which an unconfirmed result can be given out verbally and acted upon. The clinicians must be informed that the result is provisional.

It may be necessary to give therapy to the baby, and to the mother if not yet delivered, to reduce the risk of vertical transmission before confirmatory tests are completed. This depends on how long the confirmatory results will take to obtain so an individual risk assessment must be performed. This would consider the risks of delaying treatment if results will not be available within a few hours.

For example, if the screening test gave a result that was highly likely to represent a true positive and confirmation could not be completed the same day, therapy is likely to be indicated while awaiting the confirmatory results. If the result of the screening test is equivocal or a low positive and the laboratory experience of this assay is that such initial results are not confirmed and the confirmatory results can be obtained the same day, then risk of delaying treatment until the confirmed result is available may be regarded as acceptable.

The time to obtain confirmatory results could be hours or several days as it will be affected by whether the local laboratory performs the confirmatory tests in-house or sends away to a reference laboratory. Even with in-house testing, it may not be possible to confirm the screening result for 24 to 48 hours. This depends on when the initial ‘detected’ result is generated in relation to when confirmatory tests can be performed (which is rarely 24 hours a day and may be dependent on availability of laboratory staff with expertise in the relevant assays).

The reporting of the results on the laboratory IT system must wait until all confirmatory tests are completed. Once completed, laboratories must directly inform the screening team or specialist midwife of the results as outlined above.

The initial screening tests must be performed as soon as possible, ideally on sample receipt but within a maximum of 24 hours. This handbook does not specify the turnaround time for confirmatory testing, if required, but this must be expedited and local standard operating procedures must specify how this will be achieved.

7.7 Prompt testing for ‘late bookers’

Make sure tests are performed promptly for all women who are ‘late bookers’.

Women who book antenatal care late (over 20 weeks gestation) have a higher risk of adverse outcomes including pre-term delivery. The need for urgent screening is less acute than for unbooked women presenting in labour but must be performed promptly, either on the day of receipt or next working day if received out of hours.

7.8 Identifying and recording inadequate samples and requesting and receiving repeat samples

A sample is regarded as inadequate if:

• the blood sample has insufficient blood to perform all tests
• the blood sample is in the wrong tube or is lysed or contaminated
• the sample label and/or request do not have all required data fields completed accurately

Local agreements between the laboratory and maternity service must be in place to facilitate communication and timely management of:

• incomplete information on the request form or blood sample with no accompanying form
• requests for repeat samples for inadequate or insufficient screening samples
• requirement for repeat samples due to laboratory non-conformity (repeat sample must be requested as soon as non-conformity is identified by notifying the screening team and testing of the new sample must be expedited)

7.9 Monitoring result turnaround times

Make sure that the turnaround time for results (target by or before 8 working days) for each infection are monitored monthly.

Turnaround times must be reviewed at departmental quality meetings (this can be by exception) and at management reviews.

Local agreements must be in place to report the turnaround times to the local screening team every 3 months.

7.10 Antenatal screening audit

The laboratory must include in its laboratory audit schedule an annual vertical audit of an antenatal screening sample.

The audit should randomly select a confirmed screen positive sample. A different infection should be selected each year so that over a 3-year cycle confirmed screen positive samples for HIV, hepatitis B and syphilis samples are audited.

The audit should include arrival and receipt of the antenatal screening sample at the laboratory, and communication of a confirmed screen positive result by clinical services.

8. Technical requirements

Laboratories must be satisfied that they understand the capabilities and limitations of their assays. The equipment and protocol chosen must fulfil the requirements of the screening programme and demonstrate suitable performance on EQA.

8.1 All conditions

Screening test

The laboratory must adopt the screening algorithms and protocols as defined by the national screening programme. These define the conditions to be tested and the analytical methods that must be used, together with any national action limits and the diagnostic sensitivity and specificity that must be achieved. The laboratory must submit its assays on the UKAS accreditation category (AC) form and be UKAS accredited for those assays.

Point of care tests must not be used, either as the initial screen or as part of any confirmatory testing. The laboratory handbook algorithms largely follow the UK standards for microbiology investigations and these do not include point of care tests. The use of a dried blood spot or oral fluid sample as an alternative to a venous blood sample in needle phobic women is outside the scope of the IDPS pathway.

Confirmatory testing

A rapid service for confirmatory testing must be provided to avoid delays in reporting and to meet the standard for turnaround times. Confirmed positive screening tests must be reported directly to the maternity services by or before 8 days of receipt of the sample in the screening laboratory (see standard IDPS-S04). This is to enable prompt recall of the woman for discussion of the results and referral to specialist services.

Sample storage

When the screening tests are complete an aliquot (a suggested minimum volume of 300 microliters) from the screening sample must be stored frozen at a maximum temperature of -20 degrees Celsius for at least 2 years.

In cases where there is insufficient volume to store an aliquot of the screening sample then a local process must be in place to document, monitor and manage this, for example by requesting a repeat sample and informing the women of the reason. Laboratories must consider reporting the proportion of samples where it is not possible to store an aliquot of the screening sample to maternity services.

Reporting negative screens (‘negative now’)

The majority of screening samples will be negative or ‘not detected’ for all 3 infections. The phrase ‘negative now’ is being used to emphasise to women and those involved in her care that although she has tested negative in her antenatal screening sample, infection can be acquired at any time during pregnancy, after screening. Retesting may be appropriate at any stage if the woman was at risk of exposure and/or has a relevant clinical presentation. When screening is negative for all 3 infections laboratories are asked to add the following comment to reports in order to reinforce this message:

‘Negative at time the screening sample was taken. Please offer testing at any time in pregnancy if the woman has a new exposure risk or develops relevant symptoms’.

Repeat samples for confirmation

Where the woman is newly diagnosed with a confirmed positive result for HIV, hepatitis B or syphilis a repeat or confirmatory serology sample is required to confirm the positive status of the initial screening sample. Maternity units are specifically instructed to refer to specialist services on the basis of the confirmed positive result on the screening sample (except where results are inconclusive and a repeat sample is requested) and not to wait for the results on the confirmatory sample. The confirmatory sample may be sent from specialist services depending on local arrangements.

8.2 HIV

The screening test for HIV is an enzyme immunoassay (EIA) or chemiluminescent immunoassay (CLIA) or an alternative immunoassay of equivalent analytical sensitivity that detects HIV-1 antibodies, HIV-1 p24 antigen and HIV-2 antibodies. This will be a combined antigen and antibody assay and is referred to in the flowchart below and reporting table as an antigen/antibody (Ag/Ab) assay.

Assays must have a high sensitivity and specificity and be able to detect all the major HIV-1 subtypes, HIV-1 group O and HIV-2.

A cut-off value for the assay to differentiate between the negative (or not detected) and positive (or detected) samples is defined and provided in the manufacturer’s instructions.

The screening laboratory must review the assays regularly to make sure that they are able to detect new variants and that the manufacturer confirms consistent sensitivity and specificity across all subtypes.

By including HIV-1 p24 antigen detection, the Ag/Ab assays minimise the window period as infection can be detected before a woman has mounted an antibody response. These assays will detect 99% of HIV infections by 45 days post exposure.

Samples producing a negative result in the screening assay can be reported without additional testing.

Flowchart for HIV screening and confirmation

An accessible text-only version of this flowchart is also available.

Notes to support use of the flowchart

Laboratories see HIV-2 very rarely. If HIV-2 antibody is detected as a new diagnosis, refer to a reference laboratory that undertakes HIV-2 specific serology and HIV-2 RNA testing for confirmation.

HIV 1 and 2 coinfections are incredibly rare and results must be discussed with the reference laboratory.

Where the screening Ag/Ab assay is detected but the second Ag/Ab and typing assays are not detected, some laboratories may have sufficient test performance data and knowledge of pre-test probability to be able to report these assay results as HIV Ag/Ab not detected on the initial sample, with no requirement for a repeat sample. The screening test reactivity would need to be below the locally defined threshold. There is no national consensus on this.

The possibility of early infection is supported if one or both Ag/Ab assays used gives separate results for p24 antigen and HIV antibody and the p24 antigen is positive and antibody negative.

Most HIV viral load assays are not validated for serum samples and unless the laboratory has done its own in-house validation a separate EDTA sample will be required. Original sample volume may be inadequate for viral load testing even if validated. However, reflex testing from the screening sample can be performed if these concerns do not apply.

If the laboratory encounters any combination of results not covered by the flow chart or reporting tables it is recommended to seek advice from a reference laboratory.

Confirmation of a positive screening test result

Confirmatory assays outlined below must be performed on all screening samples that are positive (detected) or equivocal in the screening assay. With the exception of urgent testing (see section 7.6), results on such samples must not be reported until the confirmatory tests on the initial screening sample are performed and a conclusion reached about the full HIV results. Interim results must not be reported as they can cause confusion.

The requirement for 2 further independent assays is to confirm that the reactivity is specific for HIV and to reduce the possibility of non-specific reactions giving false positive results.

One of the confirmatory tests must be an alternative CE marked Ag/Ab assay and the other a typing assay that differentiates between HIV-1/HIV-2 antibodies. At least one assay must be performed from the clot/primary collection tube which must be checked for correct patient ID.

If a positive or equivocal result in the screening test is not substantiated by both confirmatory tests, the results are inconclusive. A repeat sample must be requested; this must be taken and sent a minimum of 2 weeks after the initial sample date.

Where the initial screening assay and the second Ag/Ab assay are both positive but the antibody only test is negative then a standalone p24 antigen assay or an HIV RNA assay must be used to identify acute HIV infection. An additional sample may be required, for example, if the initial sample is serum and plasma is required for the HIV RNA assay.

If the laboratory is using an Ag/Ab assay with integral HIV-1 and HIV-2 antibody discrimination it is not necessary to use a separate typing assay. However, if the integral typing component is negative for both HIV-1 and HIV-2 then a separate typing assay must be performed and/or a repeat sample requested.

Alternative flowchart for sequential confirmation

Some laboratories prefer to follow a sequential confirmation process, only proceeding to the typing assay if the second Ag/Ab assay is positive. If the second Ag/Ab assay is negative the typing assay is omitted and a repeat sample requested for both serology and HIV RNA a minimum of 2 weeks after the initial sample date.

Although the UK standards for microbiology investigations for HIV screening and confirmation give the option of issuing an interim report if both initial and second Ag/Ab assays are positive, the IDPS programme does not endorse this as the programme has a policy of only reporting results when all confirmatory tests are completed.

An accessible text-only version of this flowchart is also available.

Laboratories should follow the reporting guidelines given in the results table and reporting comments for HIV.

8.3 Hepatitis B

The recommended screening test for hepatitis B is an EIA or CLIA to detect hepatitis B surface antigen (HBsAg).

This must have an analytical sensitivity of at least 0.2 IU/ml and a cut-off defining a diagnostic sensitivity of over 99.9% and a diagnostic specificity of over 99.5%. The screening assay must also be able to detect a panel of the most common HBsAg escape mutations.

A cut-off value for the assay to differentiate between the negative (or not detected) and positive (or detected) samples is defined and provided in the manufacturer’s instructions.

The screening test is designed to detect women who have an acute or chronic infection with hepatitis B virus. The high sensitivity of the assays means they may also detect women who are in the early incubation phase of an infection. The further tests used to assess infectivity will identify such cases.

Samples producing a negative result in the screening assay can be reported without additional testing.

Flowchart for HBV screening and confirmation

An accessible text-only version of this flowchart is also available.

Confirmation of a positive screening test result and assessment of infectivity

The assays outlined below, with the exception of the HBV DNA assay, must be performed on all screening samples that are positive (detected) or equivocal in the screening assay. With the exception of urgent testing (see section 7.6), results on such samples must not be reported until the confirmatory tests are performed on the initial screening sample and a conclusion reached about the full HBV serology results. Interim results must not be reported as they can cause confusion.

Samples that are positive or equivocal for the HBsAg screening test must be confirmed using either a neutralisation assay or an alternative HBsAg assay with an analytical sensitivity of at least 0.2 IU/ml and able to detect immune/vaccine escape variants. Samples with very low or very high HBsAg might be unsuitable for neutralisation assays and an alternative HBsAg assay must be used. If an aliquot of serum/plasma was tested in the screening assay it is recommended that the second HBsAg is performed from the clot/primary collection tube which must be checked for correct patient identity.

Following confirmation of HBsAg status, assessment of HBV infectivity and acute infection status are undertaken on the initial sample. The assays required are anti-HBc (total), anti-HBc IgM, HBeAg and anti-HBe. These assays are performed on both newly diagnosed women and on those known to the laboratory to be living with hepatitis B.

The standard for reporting samples by or before 8 days applies to the HBV serology but not to HBV DNA. Reporting the HBV serology must not be delayed by waiting for the HBV DNA result. The initial screening sample must be tested for HBV DNA whenever possible. It is recognised that this assay may require a second sample if, for example, the initial sample is a serum sample and a plasma sample is required for HBV DNA, or if there is inadequate sample left after the other assays are completed.

Anti-HBc IgM, normally a marker of acute infection, may be detected during flares in chronic infections. It may also be non-specific. When reporting positive anti-HBc IgM results laboratory staff must look at the level of IgM and previous results (if available) in order to minimise misclassification. It is useful to determine if the raised core IgM is due to a flare or acute HBV as HBIG would not be indicated for a flare unless there were other factors such as HBeAg detected and/or if HBV DNA level is ≥ 1x10⁶ IU/ml and/or birth weight under 1.5 kg.

Acute HBV and flares can usually be distinguished:

• if the patient is previously known to be living with HBV the raised anti-HBc IgM will be due to a flare or will be non-specific
• during a flare the anti-HBc IgM will be detected but at a relatively low level and the anti-HBc (total) will be high
• in acute HBV the level of anti-HBc IgM will be high and the anti-HBc (total) will be low

If the clinical picture is unclear it is recommended to review the results after repeat serology, HBV DNA and alanine aminotransferase (ALT). If necessary, results can be discussed with the local reference laboratory.

Tests for the markers anti-HBc (total), anti-HBc IgM, HBeAg, anti-HBe and measurement of viral load enable appropriate triage of all women with new confirmed hepatitis B and women with known hepatitis B. This will also inform the ordering and coordination of the infant hepatitis B immunoglobulin (HBIG) (IDPS-S06 and IDPS-S07) and use of antiviral drugs in the third trimester.

HBV anti-HBc avidity testing is not reliable in samples from pregnant women and is not recommended. Laboratories should make sure that they have sufficient data to establish what reactivity in their anti-HBc IgM assay can be regarded as low level or high level.

Laboratories should follow the reporting guidelines given in the results table and reporting comments for hepatitis B.

8.4 Syphilis

The screening test for syphilis is an EIA or CLIA or an alternative immunoassay of equivalent analytical sensitivity that detects treponemal IgG and IgM antibodies.

A cut-off value for the assay to differentiate between the negative (or not detected) and positive (or detected) results is defined and provided in the manufacturer’s instructions. Some, but not all assays, include an equivocal range.

These first line serological screening tests rely on a woman having mounted an antibody response to their infection and may be insensitive in very early treponemal infection. This means the result may be a false negative.

Samples producing a negative result in the screening assay can be reported without additional testing.

Flowchart for syphilis screening

An accessible text-only version of this flowchart is also available.

Confirmation of a positive screening test result

The confirmatory assays outlined below must be performed on all screening samples that are positive (detected) or equivocal in the screening assay. With the exception of urgent testing (see section 7.6), results on such samples must not be reported until the confirmatory tests are performed on the initial screening sample and a conclusion reached about the full syphilis results. Interim results must not be reported as they can cause confusion.

A second treponemal test, treponema pallidum particle agglutination (TPPA) or treponema pallidum haemagglutination (TPHA) assay, must be performed as a confirmatory test on the initial screening sample. These assays are sensitive and specific and a combination of a positive result with both EIA/CLIA and TPPA/TPHA gives a strong likelihood of past or current treponemal infection.

In addition, a rapid plasma reagin (RPR) titre must be performed to assess the activity of infection. RPR is a non-treponemal test. A number of conditions, including pregnancy, can cause biological false positive results.

At least one assay must be performed from the clot/primary collection tube which must be checked for correct patient ID.

A positive or equivocal result with EIA which is reproducible, but negative with TPPA/TPHA and RPR is an inconclusive result and may be due to a false positive EIA, very early infection, or past infection with loss of TPPA/TPHA. A second sample must be requested to be sent a minimum of 2 weeks after the initial sample to look for the development of antibodies in early infection. False positive results on the initial screening test may be caused by cross-reacting antibodies in the woman’s blood. In low prevalence populations, such as pregnant women in the UK, most initial screen reactive results will be false positive results.

A positive or equivocal result with EIA, negative TPPA/TPHA but positive RPR is an unusual pattern of results. First make sure that the results can be reproduced and if they do so then a second sample must be requested to be sent 2 weeks after the initial sample. If the repeat sample produces the same results send both samples to the reference laboratory and delay reporting until receipt of the reference laboratory report.

Option to use a second screening assay

Some laboratories have a second platform on which they can run an EIA or CLIA that is different from the primary screening assay but of equivalent sensitivity. The laboratory can choose to use this second EIA/CLIA assay when the screening assay is positive or equivocal but the TPPA/TPHA is negative. Samples that are negative by both TPPA/TPHA and the second EIA/CLIA can be reported as ‘not detected’. Samples positive in the second EIA/CLIA would then require RPR.

Flowchart when using a second screening assay

An accessible text-only version of this flowchart is also available.

Laboratories should follow the reporting guidelines given in the results table and reporting comments for syphilis.

9. Screening safety incidents

A screening safety incident is any unintended or unexpected incident or incidents that could have or did lead to harm to one or more persons who are eligible for NHS screening. This also applies to staff working in the screening programmes.

A screening safety incident can affect populations as well as individuals. It is an actual or possible failure in the screening pathway.

Providers will comply with the national guidance for the management of safety concerns and incidents in screening programmes and NHS England’s guidance for managing safety incidents in NHS screening programmes.

Laboratory services are expected to inform the Medicines and Healthcare Products Regulatory Agency (MHRA) of any adverse incidents.

This latest revision of the laboratory handbook considers not only the analysis of these screening safety incidents involving the laboratory but also the screening safety incidents that did not occur in the laboratory.

We have used the lessons learned from IDPS safety incidents and serious incidents to revise this handbook. In addition, queries from SQAS, UKAS reports and the screening helpdesk were considered.