2.3. Concurrent use of initial diagnostic tests for diagnosis of TB in People living with HIV and children

There are significant burdens of tuberculosis in people living with HIV and children, particularly in low- and middle-income countries (LMICs). Persons living with HIV are at substantially higher risk of developing TB disease due to immunosuppression, with TB being a leading cause of death among this population. Children, especially those under five, are at high risk of progression from TB infection to TB disease and rapid disease progression and often present with broad respiratory symptoms, which complicate diagnosis and increase morbidity and mortality if not promptly treated. Addressing TB in these at-risk populations requires concerted efforts that account for their unique clinical presentations and diagnostic needs.

Diagnosing TB in persons living with HIV and children is challenging, particularly because of unspecific clinical presentations and often low and varying numbers of mycobacteria in their samples that lower the sensitivity of existing diagnostic tests. Furthermore, children and people living with HIV with advanced immunosuppression may be unable to provide sputum samples and can have disseminated TB, which is challenging to confirm with laboratory methods. To, in part address this challenge, WHO recommends the use of stool to aid in laboratory confirmation of TB in children, and the use of urine to aid in the confirmation of TB in persons living with HIV. However, even highly sensitive tests for TB diagnosis, such as LC-aNAATs, can miss TB in these groups. There is therefore a need for improved diagnostic approaches to accurately confirm TB in these higher-risk populations to ensure early and effective treatment.

Tests based on the detection of the lipoarabinomannan (LAM) antigen are biomarker-based tests that may be used on urine at the point of care for TB detection. The currently available urinary LAM assay is rapid (<1 hour to result) but has suboptimal sensitivity and is therefore not suitable as general diagnostic tests for TB. However, unlike traditional diagnostic methods, it demonstrates improved sensitivity for the diagnosis of TB among individuals coinfected with HIV. The estimated sensitivity is even greater in patients with low CD4 cell counts. The lateral flow urine LAM assay (LF-LAM) strip-test – the Abbott/Alere Determine TB LAM Ag (USA), hereafter referred to as LF-LAM – is currently the only commercially available urinary LAM test.

Using concurrent¹⁰ testing of different sample types offers a promising approach that considers the diagnostic testing barriers for HIV-positive adults and adolescents, HIV-positive children, and children without HIV or for whom HIV status is unknown. For instance, testing of sputum and stool during the same visit, when feasible, using LC-aNAATs increases the likelihood of detecting TB in children who may have scant bacilli in respiratory samples alone. Similarly, for persons living with HIV, testing of sputum and urine during the same visit, when sputum can be produced, using LC-aNAATs and LF-LAM increases the likelihood of detecting TB with a rapid point-of-care result while also ensuring detection of rifampicin resistance. This concurrent testing approach builds on the prior recommendation for LF-LAM test use among eligible persons living with HIV, which underscored the need for mWRD testing of available respiratory samples to support universal patient access to resistance testing services.

Implementing a diagnostic approach that includes concurrent sample testing could simplify diagnostic processes, shorten the patient journey, and improve TB detection rates and health outcomes for these at-risk populations. At the same time, the inability to collect one or more specimens at the same initial visit, or lack of one of the two test types should not delay testing of available specimens and tests, but instead trigger specimen collection and testing as soon as possible.

The following three scenarios of recommendations:

LC-aNAAT on respiratory samples and urine LF-LAM among adults and adolescents living with HIV
LC-aNAAT on respiratory samples and stool in children
LC-aNAAT on respiratory samples and stool, as well as urinary LF-LAM among children with HIV

These recommendations should be implemented within recommendations for the comprehensive diagnosis and management of persons living with HIV and children.

2.3.1 Concurrent use of tests in people living with HIV

Recommendations

Remarks

Serious illness in people living with HIV is defined based on any of the following symptoms: respiratory rate ≥30 breaths per minute, temperature ≥39 °C, heart rate ≥120 beats per minute or unable to walk unaided.
Advanced HIV disease is defined in people living with HIV who have a CD4 cell count of <200 cells/mm³ or presenting with a WHO Stage 3/4 AIDS-defining illness.
This concurrent testing recommendation supersedes prior guidance on using LF-LAM for people living with HIV and the use of a single molecular test for diagnosis of TB in this group.
This recommendation is strong despite the low certainty of evidence because the findings indicate large desirable effects (i.e. rapid and accurate diagnosis of TB in a highly vulnerable population – people living with HIV – in whom diagnosing TB is often challenging) over small undesirable effects (i.e. negative consequences of this testing strategy).
The LC-aNAAT products for which eligible data met the class-based performance criteria for this recommendation were Xpert MTB/RIF Ultra and Truenat MTB Plus. Data for performance of Truenat MTB Plus and MTB-RIF Dx were only available for testing among persons living with HIV without concurrent LF-LAM testing.

Justification and evidence

In a 2016 Cochrane systematic review of the diagnostic accuracy of LF-LAM, sensitivity increased by 13% when combining LF-LAM and sputum Xpert MTB/RIF, compared with sputum Xpert alone, while the specificity decreased by 4%. However, results were based on only a few studies, and analyses were restricted to participants able to produce sputum.

Incremental diagnostic accuracy

In 2023, WHO commissioned a series of systematic reviews to evaluate the incremental diagnostic accuracy¹¹ of concurrent use of either two different tests – LC-aNAAT on respiratory samples and LF-LAM on urine among people living with HIV – or the same test on two samples (LC-aNAAT on respiratory and stool samples) in children, or alternatively LC-aNAAT on respiratory and stool samples along with LF-LAM on urine among children with HIV.

What is the incremental diagnostic accuracy of concurrent use of respiratory LC-aNAATs and LF-LAM on urine for diagnosis of TB disease in adults and adolescents with HIV who present with presumptive TB, compared with any of the tests alone?

Of 31 studies, 27 evaluated diagnostic accuracy against an MRS, and 23 against a CRS, with 20 studies evaluating accuracy against both reference standards.

A total of 27 studies (12 651 participants, including 2368 [18.7%] with TB) compared the accuracy of the concurrent use of LC-aNAAT on a respiratory sample and LF-LAM versus each of the tests alone, using an MRS. The pooled differences in sensitivity and specificity between concurrent testing versus LC-aNAAT alone were 6.7% (95% credible interval [CrI]: 3.8 to 10.7; 95% prediction interval [PI]: 0.6 to 45.9) and –6.8% (95% CrI: –9.5 to –4.7; 95% PI: –32.8 to –6.8), respectively (Fig. 2.3.1.1). Certainty of evidence was low for both sensitivity and specificity.

A total of 23 studies (11 109 participants, including 3723 [33.5%] with TB) compared the accuracy of the concurrent use of LC-aNAAT and LF-LAM versus LC-aNAAT alone, using a CRS. The pooled differences in sensitivity and specificity between concurrent testing versus LC-aNAAT alone were 16.0% (95% CrI: 10.7 to 22.9; 95% PI: 2.3 to 60.3) and –3.5% (95% CrI: –6.6 to –1.7; 95% PI: –47.2 to –0.1), respectively (Fig. 2.3.1.1). Certainty of evidence was low for sensitivity and very low for specificity.

Fig. 2.3.1.1. Forest plot of pooled differences in sensitivity and specificity (all studies combined) by index test: LF-LAM, LC-aNAAT and their concurrent useᵃ

CrI: credible interval; CRS: composite reference standard; LC-aNAAT: low-complexity automated nucleic acid amplification test; LF-LAM: lateral flow urine lipoarabinomannan assay; MRS: microbiological reference standard; TB: tuberculosis.

^a The diamonds represent the pooled sensitivity and specificity, and the black horizontal line its 95% CrI. The pooled difference in sensitivity and specificity between concurrent testing and LC-aNAAT alone is indicated by a line connecting two diamonds. This pooled difference may not correspond to the difference between the pooled single test accuracy estimates (see Web Annex B.8).

In addition to diagnostic accuracy, clinical outcome data on mortality, time to diagnosis and time to treatment were assessed. Data on cure and loss to follow-up were not assessed due to a lack of data. The data from three studies indicated that an intervention including LC-aNAAT on respiratory samples and LF-LAM on urine in adult inpatients with HIV was associated with slightly reduced 8-week mortality (risk ratio: 0.93; 95% CI: 0.74–1.17). The adjusted hazard ratio of time to diagnosis in adult inpatients with HIV was 1.55 (95% CI: 1.29–1.87). This means that participants in the intervention groups (i.e. those undergoing concurrent LC-aNAAT on respiratory samples and LF-LAM on urine) were 1.55 times more likely to be diagnosed with TB within fewer days (relative reduction of 2 days and 1 day to same-day) than those in the control group. The pooled risk ratio of adult inpatients with HIV diagnosed with TB was 1.56 (95% CI: 1.29–1.88), indicating that the intervention group had 1.56 times the risk of being diagnosed with TB (either microbiologically confirmed or clinically diagnosed) compared with the standard of care, which included LC-aNAAT on sputum alone. The pooled risk ratio of adult inpatients with HIV with a bacteriologically confirmed TB diagnosis was 3.06 (95% CI: 1.82– 5.16), indicating that the intervention group had three times the risk of being microbiologically confirmed with TB compared with the standard of care. Finally, the pooled risk ratio of adult inpatients with HIV treated for TB was 1.47 (95% CI: 1.25–1.73), indicating that the intervention group had 1.47 times the likelihood of being treated for TB, compared with the standard of care.

Single sample testing in people living with HIV compared with the MRS

Should LC-aNAATs on respiratory samples be used to diagnose pulmonary TB in PLHIV (adults and adolescents) with signs and symptoms or screened positive for pulmonary TB, against a microbiological reference standard?

Twelve studies (2016 participants) evaluated sputum specimens from people living with HIV (Fig. 2.3.1.2). The sensitivities ranged between 54% and 100% and the specificities between 78% and 100%. The summary sensitivity (95% CI) was 87.4% (83.8 to 90.3) and the summary specificity was 95.2% (92.7 to 96.9). The certainty of evidence for both sensitivity and specificity were graded as “High”.

Fig. 2.3.1.2 Forest plot of LC-aNAAT sensitivity and specificity for detection of pulmonary TB in PLHIV using a microbiological reference standard

Studies are sorted on the plot by assay and sensitivity (low to high). FN: false negative; FP: false positive; TN: true negative; TP: true positive.

Cost–effectiveness analysis

To date, evidence of cost–effectiveness for concurrent testing is limited. Several studies have assessed Xpert MTB/RIF with LF-LAM for diagnosing TB among people living with HIV. These studies have shown that concurrent testing is likely to increase the life expectancy of people living with HIV and be cost effective compared with using Xpert MTB/RIF in sputum samples alone. Fekuda et al. evaluated the cost–effectiveness of concurrently using Xpert Ultra and LF-LAM among people living with HIV and concluded that concurrent testing is the preferred cost-effective strategy. Previous cost–effectiveness analyses primarily focused on Xpert MTB/ RIF or Xpert Ultra, leaving a gap in evidence regarding the other technologies that may meet the LC-aNAAT class criteria. For details of particular studies see Web Annex B.9.

In preparation for the GDG meeting in May 2024, WHO commissioned a study to assess the cost– effectiveness of using LC-aNAATs (including Xpert Ultra, Truenat and other novel LC-aNAATs in the development pipeline) for the detection of TB when used concurrently among people living with HIV and children, including children with HIV, across two different country settings (Malawi and the Philippines). An objective of the study was to assess the cost–effectiveness of concurrent use of LC-aNAAT on respiratory samples and LF-LAM on urine for TB diagnosis and rifampicin-resistance detection among adult people living with HIV with presumptive TB, compared with a single LC-aNAAT on respiratory samples alone.

In the hypothetical model, a cohort of people living with HIV with signs and symptoms of TB progressed through a decision analytical framework. In the intervention arm, TB diagnosis involved the concurrent use of LC-aNAAT on respiratory samples and LF-LAM on urine, whereas the comparator arm exclusively used LC-aNAAT on respiratory specimens. The probability of being able to provide a respiratory sample was considered, and testing was carried out, either on both respiratory and urine samples concurrently or solely on urine. In both intervention and comparator arms, participants not diagnosed through the diagnostic strategy had the opportunity for clinical diagnosis. People with bacteriologically confirmed TB underwent DST for rifampicin and began either drug-susceptible TB or DR-TB treatment, depending on the DST result. All individuals were followed over time, including those with false negative or false positive diagnostic results, to account for unnecessary treatment or additional mortality due to missed diagnoses.

The cost–effectiveness results of concurrent use of LC-aNAAT with LF-LAM among people living with HIV, when used in the emblematic settings of Malawi and the Philippines, are shown in Table 2.3.1.1 In Malawi, the average cost of implementing an LC-aNAAT on a respiratory sample was US$ 276, with a corresponding average DALY of 2.44. When used concurrently with LF-LAM, the average cost rose to US$ 298, while the average DALY decreased to 1.93. The resulting incremental cost per DALY averted was US$ 42, with a 95% uncertainty range (UR) of US$ 18 to US$ 345. Similarly, in the Philippines, LC-aNAAT on a respiratory sample had an average cost of US$ 220, with an average DALY of 2.78, whereas concurrent use with LF-LAM incurred an average cost of US$ 238 and an average DALY of 2.13. The incremental cost per DALY averted was US$ 28 (95% UR: 12–249).

Table 2.3.1.1 Cost–effectiveness analysis of concurrent use of LC-aNAAT and LF-LAM among people living with HIV in Malawi and the Philippines

DALY: disability-adjusted life year; HIV: human immunodeficiency virus; ICER: incremental cost–effectiveness ratio; LC-aNAAT: low-complexity automated nucleic acid amplification test; LF-LAM: lateral flow urine lipoarabinomannan assay; UR: uncertainty range.

More information on the cost–effectiveness analysis of concurrent use of tests in people living with HIV is available in Web Annex B.9.

User perspective

This section deals with the following question:

Are there implications for user preferences and values, equity, acceptability, feasibility and human rights from the implementation of a concurrent testing approach (LC-aNAATs + LF-LAM)?

The GDG assessed whether concurrent testing of multiple samples would increase the diagnostic accuracy (i.e. the benefit to patients or the programme in terms of finding more people with TB). Three PICO questions concerned the different concurrent sample combinations for specific groups facing challenges from reliance on respiratory samples alone (children and people living with HIV). One question focused on the concurrent use of LC-aNAAT on a respiratory sample and LF-LAM on urine for the diagnosis of TB in people living with HIV.

User preferences and values

As important outcomes of the diagnostic test, people in high TB burden settings value:

getting an accurate diagnosis and reaching diagnostic closure (finally knowing “what is wrong with me”);
avoiding diagnostic delays, as they exacerbate existing financial hardships and emotional and physical suffering and make people feel guilty for infecting others (especially children);
having accessible facilities; and
reducing diagnosis-associated costs (e.g. travel, missing work).

More details on patient-important outcomes are available in Web Annex B.10.

Equity

Concurrent specimen testing was not practiced in the interview study countries. However, it was believed to improve access to care by minimizing repeat visits and loss to follow-up. According to the interview study respondents, using non-sputum specimens has the potential to improve access to care, especially with a test that can be performed at all levels of the health care system. Challenges with producing a sufficient quality and quantity of sputum are well documented and can lead to repeat testing or false results.

Acceptability

Based on the results of the interview study, LF-LAM is being used inconsistently for people living with HIV and only for very ill patients who cannot produce sputum. Our results are in accordance with published literature on LF-LAM.

Prior research on user perspectives on LF-LAM showed that it is generally described as acceptable by key stakeholders, due to its fast turnaround time, ease of use (lack of technical expertise required), low or no maintenance and equipment required, and urine being more accessible and less stigmatized than sputum. LF-LAM is deemed particularly acceptable when used in combination with other tests and clinical considerations. As the sensitivity of LF-LAM is especially low where the pretest probability is low, participants commented that it should not be used as a standalone test but should instead be used in combination with other tests, and that the results should be interpreted by a doctor considering the full clinical context, rather than being considered in isolation.

Feasibility

Interview study findings highlighted that the benefits of LF-LAM are crucially dependent on how several feasibility challenges are addressed.

Hygienic, safe and private sanitary facilities with running water are necessary for LF-LAM implementation at a testing site, but they are not always available, particularly in rural areas. Investments in staffing and sanitary facilities are required.
Not everybody can spontaneously produce or collect urine samples. This can be the case, for example, when the patient is too ill or septic or has to be catheterized because collecting urine samples from diapers is impossible, or if the hospital has no clean, private space to produce urine.
Visibility of faint results and result interpretation can be problematic. Comprehensive health care worker training in test interpretation (including mandatory use of the reference reading card, where appropriate) is crucial to ensure accurate result interpretation for clinical action.
The need for CD4 cell count results to select people for the test is problematic because these are not always immediately available. To facilitate implementation and benefit a wider range of individuals, eliminating the CD4 cell count as an eligibility criterion for people living with HIV should be considered.
In a hospital setting, bedside testing may violate patient confidentiality.
Results must be captured in a standardized way that feeds into facility and NTP reporting systems.
Quality assurance schemes need to be rolled out, and external quality controls need to be made available, to ensure tests and testing processes are quality controlled.

Concurrent testing needs to be framed as a more efficient way of working (i.e. testing two samples concurrently during the same visit, instead of testing one sample during each of two separate visits) that also allows increasing access and reducing costs for patients. According to a laboratory manager, this framing of the benefits outweighing the additional workload, and potentially resulting in reduced work in the long run, will be critical to avoid concurrent testing being perceived as additional work for already overburdened health care workers (see Web Annex B.10).

Prior investments made in frontrunner technologies, donor preferences, limited health systems thinking and unnecessary competition between manufacturers all pose challenges to policy adoption and implementation of novel molecular diagnostics. In addition, national in-country health technology and cost-efficacy assessments can delay decisions to implement newer technologies and diagnostic strategies using different samples (see Web Annex B.10).

Implementation considerations

Global and national HIV and TB programmes need to communicate regularly and clearly, indicating responsibilities for concurrent testing for people living with HIV.
Concurrent testing maximizes diagnostic opportunity and accuracy of case detection, is a more efficient way to address the needs of this population and is preferred even if the testing workload may increase.
A positive result on either test is sufficient to confirm TB diagnosis.
Patient loss to follow-up for the second test result should be monitored and prevented. Patients should be provided with information to understand the concurrent testing approach and the need for follow-up.
The LF-LAM performed in point-of-care settings may be the first positive result and is sufficient to make the initial diagnosis. A respiratory sample is still required for rifampicinresistance detection, and is also required when the LF-LAM result is negative.
Where LF-LAM is not available for testing of people living with HIV, efforts should be made to ensure access to testing.
LF-LAM does not differentiate Mtb from other mycobacterial species. However, the LAM antigen detected in a clinical sample in TB endemic areas is most likely attributable to Mtb.
When LF-LAM results are consistently positive, without positive LC-aNAAT results, investigation of the quality of testing and local epidemiology of non-tuberculosis mycobacteria and extrapulmonary TB in the tested population is warranted to understand the difference.
Interpreting bands on the LF-LAM test strip should be performed using the manufacturer’s reading card to minimize incorrect results.
LF-LAM test strips must be stored according to the manufacturer’s instructions (e.g. between 2 and 30 °C) in sealed bags and not used after expiration.
Infrastructure to collect a urine sample privately should be available. Patients should be instructed how to properly and sanitarily collect a urine sample to minimize contamination and prevent false positive results.
Trained staff will be required to perform the LF-LAM test at the point of care.
As with all WHO-recommended TB diagnostics, quality assurance programmes and quality controls for both tests are required.
LF-LAM is designed to detect mycobacterial LAM antigen in human urine. Other samples (e.g. sputum, serum, plasma, CSF and other body fluids) or pooled urine specimens should not be used.

Monitoring and evaluation

Monitor simultaneous specimen collection and turnaround time for the test results in a concurrent testing approach.
Monitor patient access to, and loss to follow-up from, a second test in a concurrent testing approach.
Monitor patient access to, and loss to follow-up from, follow-on DST among those with a positive LF-LAM result but a negative LC-aNAAT result.
Monitor trends in the discordance rate between the LF-LAM and LC-aNAAT results. If these differences vary from other local or regional patterns, or if the trends change, further investigation is required and outcomes should be tracked for recurrence over time.

Research priorities

Conduct more rigorous studies with higher quality reference standards, including multiple specimen types and extrapulmonary samples, to improve confidence in specificity estimates.
Gather evidence on the impact of concurrent testing on TB treatment initiation and mortality.
Determine training, competency and quality assessment needs by setting and by cadre of staff (i.e. health care worker, laboratory technician or clinical staff).
Perform country-specific cost–effectiveness and cost–benefit analyses of the concurrent testing approaches or sequential testing approaches in different programmatic settings.
Develop and apply standardized methods for assessment of costs and cost–effectiveness, to improve comparability and scope of economic evidence.
Perform operational research on availability, requirements and best practices for the pointof-care set-up: private specimen collection facility, tabletop space for testing samples, and reporting system (preferably digital) for entry of results, with linkages to existing information management systems (i.e. health and laboratory information management systems).

2.3.2 Concurrent use of tests in children without HIV or with unknown HIV status