Diagnostic Tests for Childhood Tuberculosis

Over the past decade, there has been significant progress in developing new diagnostic tools for childhood tuberculosis (TB).1 However, there are still many unanswered questions, and the search for accurate diagnostic tests is far from over. This review provides an overview of existing immunological and microbiological tests for TB, with particular focus on their strengths and limitations, and discusses novel methods and directions for future research. THE EPIDEMIOLOGY OF CHILDHOOD TUBERCULOSIS The true global incidence and prevalence of childhood TB remain uncertain,2 largely because microbiological confirmation of active TB (also called TB disease) is not obtained in the majority of children for a number of reasons. First, children typically have paucibacillary disease, which hampers detection of Mycobacterium tuberculosis. Second, respiratory samples are difficult to obtain in young children and few healthcare facilities are set up to obtain induced sputum samples in routine practice, which have been shown to increase detection yields.3 Consequently, worldwide the majority of children with active TB are started on antimycobacterial treatment based on history, symptoms and clinical signs—with or without supporting radiological findings—alone. Until recently, the World Health Organization (WHO) and other public health agencies made little effort to capture children with non- microbiologically confirmed active TB, as official figures for TB incidence were simply based on the number of smear- and culture-confirmed cases. The WHO Global Tuberculosis Report in 2012 was the first to include estimates for childhood TB. The latest edition (2014) includes estimates for new TB cases in children (an estimated 550,000 cases) and TB-related deaths in HIV-negative children (an estimated 80,000 cases).4 Notably, the report does not include an estimate for TB-related deaths in HIV-positive children, which is likely to eclipse the figure in HIV-negative children. However, the WHO estimates are based on detection rates in adults, and therefore likely considerably underestimate the burden of TB in children. It is therefore not surprising that a recent study based on mathematical modeling arrived at substantially higher figures, estimating the annual global incidence of active TB in children to be greater than 650,000 cases.5 ACTIVE TUBERCULOSIS VERSUS LATENT TUBERCULOSIS INFECTION Traditionally, infections with M. tuberculosis have been categorized into active TB and latent TB infection (LTBI). Patients with active TB typically have symptoms and/or signs, which depend on the site of the infection, and the infection may be confirmed by conventional (ie, microscopy or culture) or molecular (eg, PCR) microbiological methods (depending on the infection site, adequacy of the sample and the methods used). In contrast, classical dogma suggests that patients with LTBI are asymptomatic, and that their immune system is containing M. tuberculosis. Consequently, current microbiological methods are unable to detect the mycobacteria, and the diagnosis of LTBI is therefore solely based on immune-based tests that detect memory T cells (and potentially other immune cells) induced by exposure to antigens expressed by M. tuberculosis [ie, either tuberculin skin test (TST) or interferon-gamma release assay (IGRA)]. The segregation between active TB and LTBI remains useful from a clinical as well as a programmatic perspective as this distinction currently determines the treatment approach (ie, treatment with 1 or 2 antimycobacterial drugs for LTBI vs. 3 or more drugs for active TB). However, there is increasing evidence that active TB and LTBI are not discrete infection states, but rather opposite ends of a continuum. Historical data from before the advent of antimycobacterial drugs illustrate that some patients with active TB survive without treatment; more recent data show that these asymptomatic survivors maintain TB-specific immunological memory for decades.6 Thus, if tested with immune-based tests (TST or IGRA) after recovery, such individuals would currently have to be classified as “LTBI.”7 Furthermore, recent studies in both nonhuman primates and humans using newer radiological methods, such as positron emission tomography, show that disease activity can be detected in a significant proportion of individuals with a positive immune-based test who are asymptomatic, and therefore would be classified as “LTBI” according to current criteria.8,9 It is uncertain whether those individuals would convert to overt “active TB” if left untreated. This question will likely remain unanswered given that withholding anti-mycobacterial treatment under those circumstances would be unethical. In addition, several recent publications have described patients with “subclinical TB disease,” who are clinically asymptomatic, but have positive sputum cultures (with or without smear-positivity).10,11 Despite these issues, for clarity we will continue to use the terms “active TB” and “LTBI” in this review. IMMUNE-BASED TESTS AND THEIR LIMITATIONS Tuberculin Skin Test Since the early 20th century, purified protein derivative (PPD), a heterogeneous mixture of mycobacterial peptides, has been used as the test substance for the TST (also called Mantoux test). The TST is commonly used to support the presumptive diagnosis of active TB, and was the only available test for the detection of LTBI, until IGRA became commercially available in 2002. The key limitation of the TST lies in its limited specificity. False-positive results can occur as a result of prior BCG immunization or infection with nontuberculous mycobacteria (NTM), as both BCG and NTM express peptides that are present in PPD. False-negative results can also occur as a result of immunodeficiency, immunosuppression, malnutrition and errors in test administration or reading. The test requires reading after 48 to 72 hours, which is inconvenient for both healthcare providers and patients; if reading is not performed within this time window, the validity of the test result becomes questionable. Despite those limitations, the latest recommendations of the American Academy of Pediatrics Committee on Infectious Diseases state that for LTBI screening in children less than 5 years of age TST should be used in preference of IGRA.12 However, the recommendations also highlight that the combined use of TST and IGRA results in an increase in diagnostic sensitivity. An additional major limitation of the TST remains the subjectivity in the reading of the resulting induration, the extent of which determines the test result. The current standard technique for reading—the “ballpoint technique”—involves palpating for the outer edges of the induration, marking these with a ballpoint pen and measuring the diameter of the induration using a flexible ruler or a caliper. This technique is prone to considerable inaccuracy with both intra- and inter-observer variability, particularly when the induration is not circular.13 Optimization and standardization of the reading technique to achieve greater accuracy and reproducibility have recently been investigated using a variety of novel approaches, including measurement by Doppler imaging, spectrophotometry and ultrasound.14 Another significant problem with the TST are recurrent shortages of PPD. World-wide, only a small number of manufacturers produce PPD for clinical use, so supply shortages at 1 manufacturer can affect the global market. Following production problems at Evans Vaccines in 2003, the UK had to source PPD from the Statens Serum Institut (SSI) in Denmark as an ‘unlicensed medicine’.15 In 2013, there was a national shortage of PPD in the US caused by production problems at Sanofi Pasteur (Tubersol) leaving only 1 FDA-licensed product (Aplisol; JHP Pharmaceuticals).16 Later that year, 29 of 52 US jurisdictions reported a shortage of at least 1 of the 2 PPD products “to the extent that routine activities were being threatened or had been curtailed.”17 Canada also experienced a less well-publicized PPD shortage in late 2012.18 A further, Europe-wide shortage of PPD was highlighted by a recent survey of TB experts based in 23 different European countries.19 Sixty percent of these (from 14 different countries) reported a PPD shortage at the time the survey was conducted (June to July 2014). The majority of those reporting a PPD shortage were using RT23 (SSI; 81.0%); fewer reported shortages of Tubertest (Sanofi Pasteur; 9.5%), PPD Tuberculin (BulBio; 4.8%), and PPD Tuberculin (St. Petersburg Institute of Vaccines and Sera; 4.8%). Interferon-gamma Release Assays Currently, 2 commercial IGRA are available for clinical use, the QuantiFERON-TB Gold In-Tube (QFT-GIT; Cellestis/Qiagen, Carnegie, Australia) and the T-SPOT.TB assay (Oxford Immunotec, Abingdon, UK). Although IGRA are solely cleared for the diagnosis of LTBI, in clinical practice they are commonly used to support a presumptive diagnosis of active TB. Both assays rely on the detection of interferon-gamma secreted by memory T cells following stimulation with mycobacterial antigens. Both assays incorporate the relatively M. tuberculosis-specific RD1 peptides antigens early secretory antigenic target 6 (ESAT-6) and 10 kDa culture filtrate protein (CFP-10); the QFT-GIT incorporates and additional antigen, TB7.7. Based on the test principle, IGRAs likely have greater specificity than the TST and are not confounded by prior BCG immunization, as all 3 stimulatory antigens are absent from all BCG vaccine strains in current use. Also, the impact of current or previous NTM infection and/or exposure on test specificity is relatively limited, since only a small number of NTM express ESAT-6 and CFP-10.20 However, (“false”) positive IGRA results have repeatedly been reported in individuals infected with Mycobacterium kansasii, Mycobacterium marinum and Mycobacterium szulgai.21,22 Although initially heralded as a tool that has the potential to revolutionize the diagnosis of TB, there are now legions of publications highlighting the limitations of IGRA.12,23–25 First, similar to the TST, the performance of IGRA in patients with immunodeficiencies or receiving immunosuppressive treatment is overall poor (the extent of which depends on the type of the immunodeficiency and the degree of immunosuppression), and false-negative assay results remain a significant problem in these patient groups.26,27 This problem is further compounded by those patients also being at greatest risk of progression from LTBI to active TB, making the accurate identification of latent infection in those individuals crucially important.12,25 As there is no gold standard for the diagnosis of LTBI (with the TST having previously been regarded as the gold standard) the true sensitivity of IGRA for the detection of LTBI cannot be determined. Nevertheless, several robust meta-analyses on the performance of IGRA as a supportive tool for the diagnosis of active TB have shown that IGRA perform no better than the TST in this setting, reporting a pooled sensitivity of approximately 60–80% in immunocompetent individuals, and even lower pooled estimates in HIV-infected patients.28,29 There is now a considerable body of evidence that the performance of IGRA is worse in young children compared with adults.12 Although the precise underlying mechanisms remain uncertain, it is likely that incomplete immune maturation plays a significant role.30 Indeterminate IGRA results (resulting from failed negative or positive control samples), which convey no information regarding the TB infection status of the patient, are significantly more common in young children than in adults.26,30,31 One pediatric study reported that indeterminate QFT-GIT results occurred in 83 (35%) of the 237 study participants.26 However, in that study, a large number of children were immunodeficient or were receiving immunosuppressive medication. The majority of pediatric studies in countries with low HIV prevalence have reported lower rates of indeterminate IGRA results, generally ranging between 5% and 20%.32–34 In addition to young age and immunodeficiency/immunosuppression, further factors that have been found to be associated with indeterminate IGRA results include malnutrition, chronic renal disease, autoimmune conditions, malaria and co-existing helminth infections.35,36 Preanalytical sources of assay variability that can result in indeterminate assay results include delays in sample incubation and inadequate shaking/mixing of QFT-GIT tubes.37,38 Further limitations of IGRA include their relatively high cost and the need for adequate laboratory facilities, which precludes their use in many resource-limited, high TB prevalence settings. In addition, several studies have shown that the reproducibility of IGRA is suboptimal when serial testing is performed (ie, with unexplained conversions from positive to negative and vice versa).12 Furthermore, while there is a large amount of longitudinal data related to the TST and its predictive value for the development of active TB, those data in relation to IGRA, particularly in children, still remain relatively limited.25 Discordance Between TST and IGRA Results Many pediatric TB experts use the TST and IGRA in parallel with the aim of increasing sensitivity, but this can be complicated by contradictory results. The underlying mechanisms of this discordance remain uncertain. In most pediatric studies, the number of children with a TST+/IGRA− result constellation is far greater than that with a that TST+/IGRA− discordance results from prior BCG immunization or induced by exposure to and that it should be as a TST result the IGRA is as a result of greater However, the evidence to support this is There is no that BCG immunization can produce (“false”) positive TST results. However, an of data that more than with BCG in found only had a positive TST result as that was to the while only were when tested 10 years or more after with BCG immunization cannot for the large proportion of children with TST+/IGRA− discordance reported by the majority of pediatric studies, which typically between and of the study There is also no that NTM disease positive TST results in a proportion of Nevertheless, NTM disease is with most publications estimating the incidence in children to be less than 10 children NTM disease can also not for the proportion of individuals with TST+/IGRA− discordance in pediatric The that NTM exposure can memory T cells and produce TST results is and the data to support this are NTM are and the of of NTM from sources is Despite the majority of individuals in low TB prevalence countries are that NTM exposure has limited impact on TST results. The for TST+/IGRA− discordance is that the TST result is while the IGRA result is false-negative a result of the having Notably, recent data evidence that a significant proportion of individuals with TST+/IGRA− discordance are in infected with M. assays with ESAT-6 and as stimulatory and were found to be significantly higher in a of children with TST+/IGRA− discordance compared with a of children. This can not be by caused by prior BCG since ESAT-6 are expressed by of the currently used BCG vaccine In the of these in a with a TST+/IGRA− result the of latent TB cannot be Consequently, as well as other TB that children with risk factors for TB infection TB exposure or in a high TB prevalence with this result constellation should be treatment until such time that better assays that can latent infection with M. tuberculosis This into that children are at far greater risk of progression from LTBI to active TB their compared with adults, and the that with standard treatment (ie, or in with are in children. 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An additional for use of the assay in the pediatric is that the assay requires a of 3 for the The current commercially available TB tests have sensitivity and and the WHO therefore their Notably, the WHO has the development of more robust and new assays are currently in studies using a variety of methods for the detection of M. tuberculosis-specific T cells have also shown considerable from 1 study in that of T cells (ie, T cells more than 1 may the distinction between LTBI and active TB based on a test A more recent study in children the diagnostic potential of T on However, the existing data for both assays are currently limited, and of these methods into a routine diagnostic in the future their current cost and In contrast, assays based on the detection of in following stimulation with M. tuberculosis-specific antigens (ie, based on similar as IGRA) are more likely to be into the routine diagnostic setting, as this can be with and more robust A small number of studies data that these assays may also the distinction between LTBI and active A recent the potential of using for the diagnosis of The a that a sensitivity of and specificity of However, this approach currently requires and it is uncertain whether this can be for use in a routine diagnostic Assays based on the detection of in sputum or in have been in the a commercial the TB has tests generally have sensitivity to be used as but perform better in HIV-infected patients with low T assays can only be used for the diagnosis of of TB and and have been shown to have limited sensitivity The recent detection of in the of patients with TB has new this approach but it is likely that this will relatively and perform worse in individuals with paucibacillary disease the majority of Also, tests will not have the to detect TB. This review that considerable in recent the search for TB tests with better performance diagnostic tests have suboptimal sensitivity, and generally perform worse in children compared with tests should particularly aim to achieve greater sensitivity, and would be or as these samples can be obtained relatively from children. be useful in in which childhood TB is most tests should be without the need for an existing laboratory Furthermore, a test that can between LTBI and active TB is as this would to tests for LTBI should also have a better predictive value for the development of active TB, given that the predictive of both TST and IGRA are As new tests are it is crucially that their performance are in large studies in children, in of the that existing tests perform worse in this patient further is to existing diagnostic approaches, in particular the use of the assay in the routine clinical setting, impact on patient and

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