The political declaration at the first United Nations (UN) high-level meeting on tuberculosis (TB) held on 26 September 2018 included commitments by Member States to four new global targets (1). One of these targets is diagnosing and treating 40 million people with TB in the 5-year period 2018-2022. The approximate breakdown of the target is about 7 million in 2018 and about 8 million in subsequent years.
In 2019, an estimated 10 million people fell ill with tuberculosis (TB); of those 10 million people, 3 million were not reported to have been diagnosed and notified (1). The gap is proportionately wider for drug-resistant TB (DR-TB). Of the estimated 465 000 patients with multidrug-resistant TB or rifampicin-resistant TB (MDR/RR-TB), only 206 030 (44%) were diagnosed and notified. For the first time, the World Health Organization (WHO) has provided global estimates of the incidence of isoniazid resistance: in 2019, there were 1.4 million incident cases of isoniazid-resistant TB, of which 1.1 million were susceptible to rifampicin (1). Most of these people were not diagnosed with DR-TB and did not receive appropriate treatment.
WHO's End TB Strategy calls for the early diagnosis of TB and universal drug-susceptibility testing (DST), highlighting the critical role of laboratories in the post-2015 era in rapidly and accurately detecting TB and drug resistance (2). Of the 7.0 million new and relapse cases notified in 2018, 5.9 million (85%) had pulmonary TB. Of these, 55% were bacteriologically confirmed, a slight decrease from 56% in 2017 and 58% in 2013.⁵ The remaining patients were diagnosed clinically; that is, based on symptoms, abnormalities on chest radiography or suggestive histology.
Activities to strengthen TB diagnosis must be viewed in the context of recent global initiatives to "find the missing cases", and the new global target set at the UN high-level meeting on TB in September 2018. In this context, the proportion of notified cases that are bacteriologically confirmed needs to be monitored. The microbiological detection of TB is critical because it allows people to be correctly diagnosed and started on the most effective treatment regimen as early as possible. Most clinical features of TB have low specificity, which may lead to incorrect diagnoses of TB, and unnecessary TB treatment. The aim should be to increase the percentage of TB cases confirmed bacteriologically, based on scaling up the use of recommended diagnostics that are more sensitive than smear microscopy.
WHO has endorsed a range of new diagnostic technologies during the past 10 years. The amplification and detection of M. tuberculosis complex (MTBC) nucleic acids is a technology that has proven to be highly sensitive and specific.
A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence. In general, a NAT is used to detect and identify a particular species or subspecies of an organism (e.g. a virus or bacteria that acts as a pathogen in blood, tissue or urine). NATs differ from other tests in that they detect genetic materials (RNA or DNA) rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens or antibodies (or both) often requires time for the antigens or antibodies to start appearing in the bloodstream (3). As the genetic material is usually present at a low level, many NATs include a step that amplifies the genetic material (i.e. makes many copies of it) -such NATs are called nucleic acid amplification tests (NAATs). Amplification of the genetic material uses the polymerase chain reaction (PCR) method, with the standard approach requiring thermal cycling. However, some do not cycle and are operational isothermally, such as the loop-mediated isothermal amplification (LAMP) method. Amplification technologies can detect amplicons in real-time using fluorescence detectors while others require visual reading. NAATs have the added advantage of detecting specific mutations associated with resistance to selected anti-TB drugs.
The lateral flow technology detecting MTBC specific antigen in a point-of-care test format has also been endorsed for use in certain groups of presumptive TB patients. In total, four classes of technologies and 4 individual products are recommended:
The real-time PCR methods that are automated and provide an all in one solution suitable for the peripheral level are the most widely used today. These tools detect MTBC DNA and can detect mutations in the gene associated with rifampicin resistance. The available tools use software and hardware (computers) to report results, and require well-established laboratory networks and trained personnel.
LPAs are a family of DNA strip-based tests that can detect the MTBC DNA and determine its drug resistance profile. The tests do this through the pattern of binding of amplicons (DNA amplification products) to probes that target the specific parts of the MTBC genome, common resistance-associated mutations to anti-TB drugs or the corresponding wild-type DNA sequence (4). LPAs are technically more complex to perform than the Xpert MTB/RIF assay; however, they can detect resistance to a broader range of first-line and second-line agents and provide mutation specific data for common variants. Testing platforms have been designed for a reference laboratory setting and are most applicable to high TB burden countries. Results can be obtained in 5 hours (5). There are two groups of assays:
- those detecting MTBC and resistance to first-line anti-TB agents (known as first-line LPAs [FL-LPAs]) - for example, GenoType MTBDRplus v1 and v2, Genoscholar NTM+MDRTB II, GenoScholar PZA-TB; and
- those detecting resistance to second-line anti-TB agents (known as second-line LPAs [SL-LPAs]) - for example, GenoType MTBDRsl.
A third technology is based on LAMP methodology, in which target DNA is amplified at a single temperature range (in contrast to the PCR, which requires a thermocycler). Detection of amplified product is done visually, using an ultraviolet (UV) lamp, directly in the reaction tubes. The method requires only basic equipment and can be implemented at the lowest levels of the laboratory network. However, detection of mutations in resistance-associated genes is not available with the currently recommended technology.
The search for a point-of-care test (i.e. a lateral flow test detecting either MTBC antigen or antibodies to MTBC) has proven difficult. However, the mycobacterial lipoarabinomannan (LAM) antigen in urine has emerged as a good candidate. The currently available urinary LAM assays have suboptimal sensitivity and specificity and are therefore not suitable as diagnostic tests for TB in all populations. However, unlike traditional diagnostic methods, urinary LAM assays demonstrate improved sensitivity for the diagnosis of TB among individuals coinfected with HIV.
A Guideline Development Group (GDG) was convened by WHO on 7-18 December 2020 to discuss the findings of the systematic reviews on three classes of diagnostic technologies and make recommendations.
The three classes of technologies evaluated include:
- moderate complexity automated NAATs for the detection of TB and resistance to rifampicin and isoniazid;
- low complexity automated NAATs for the detection of resistance to isoniazid and second-line anti-TB agents, and
- high complexity reverse hybridization-based NAATs for the detection of pyrazinamide resistance.
The WHO assessment process for TB diagnostics currently focuses on evaluating classes of TB diagnostic technologies rather than specific products. For this guideline update, the three classes of technologies evaluated here were defined by the type of technology (e.g. automated or hybridization-based NAATs), the complexity of the test for implementation (e.g. low, moderate or high - considering the requirements of infrastructure, equipment and technical skills) and the target conditions (TB and resistance to first-line or second-line drugs, or both). The level of complexity is only one of the elements that should be considered to guide implementation; others include diagnostic accuracy, the epidemiological and geographical setting, operational aspects (e.g. turnaround times, throughput, existing infrastructure and specimen referral networks), economic aspects and qualitative aspects of acceptability, equity, and end-user values and preferences.
⁵ A bacteriologically confirmed case is one for whom a biological specimen is positive by smear microscopy, culture or WHO-recommended rapid diagnostic test.