ACERCA DE LOS ALUMNOS

At the time when I formulated my doctoral research, I identified the following aspects as the most relevant problems with currently available diagnostic approaches for paediatric PTB:

1. Microbiological tests, including smear microscopy, molecular tests and culture, have low sensitivity to detect most children with PTB, who typically have paucibacillary disease (low organism load). In order to improve microbiological detection, it is therefore usually recommended that multiple specimens be collected. This has implications for both the feasibility and the cost of investigations in resource limited settings: given that most childhood PTB occurs in children below 5 years of age, who cannot readily expectorate sputum samples, the collection of appropriate clinical (respiratory) specimens for diagnostic testing requires trained health care workers, equipment and adequate clinical and laboratory facilities resulting in significant resource requirements.

2. There has been limited research to date on the relationship between the spectrum of PTB disease in children and the performance of microbiological tests. Tests applied indiscriminately produce low numbers of confirmed PTB cases in children and high costs, both of which result in low confidence to

attempt the bacteriological confirmation of PTB in children. This phenomenon is often used to justify lack of health care expenditure on TB diagnostic testing for children in many low-resource settings.

3. As a result, health care workers in resource-limited settings usually rely on clinical algorithms to diagnose TB in children, which have high levels of variability and poor specificity 43,44_{. In many settings, a diagnosis of TB is}

missed or delayed, leading to progression of disease and even death, especially in young children, where the risk of disease progression is the highest 32,57_.

4. To date, there has been limited incentive to develop new diagnostic tests specifically tailored for children, in view of the challenge of not having a reference standard with both high sensitivity and specificity for TB disease, to serve as an adequate comparator. In addition, the perceived low public health priority of paediatric TB (although children contribute at least 10% of the estimated global TB case load), has also been a disincentive to invest in diagnostic tests for children, who typically do not transmit M.tb. As a result, new tests are developed for adults and tested on adult populations first, without specific paediatric consideration. Their current modest performance in children results in further lack of confidence to attempt diagnostic confirmation in children with presumed (clinically suspected) TB.

5. Historically, when I started my doctoral research, the data on diagnostic studies for paediatric PTB had much variability, including differences in study methodology, the spectrum of TB disease included and described, and the typical lack of long-term clinical follow-up. Published studies have often been

of variable or poor quality 58-60 _{with limited internal validity and}

generalizability. Non-evaluable results, including indeterminate/invalid results and contaminated cultures, are infrequently reported. Combining data, for example in meta-analyses, to inform policy recommendations, is also problematic given differences in study methodology 31,60_.

In 2012, a collaborative international consultation among experts in the field of paediatric TB, supported by the United States National Institutes of Health (NIH), developed consensus recommendations on clinical case definitions for paediatric PTB for application in diagnostic research 61_{, and on methodological issues for PTB}

diagnostic research in children 31_{. The aim of these guidance documents was to}

develop a more standard approach for the evaluation of new diagnostics for paediatric PTB. These clinical case definitions were revised in 2015, following early evaluations from several groups. However, the evidence base for some of the elements included in the case definitions still remained limited, for example the utility and timing of follow-up as a diagnostic criterion to confirm or exclude TB disease. The 2015 NIH PTB case definitions acknowledged that further research was urgently needed, including on the value of follow-up and response to treatment in the diagnosis of PTB in children.

Given the challenges outlined above, and the need identified by the global research community to generate high quality data to inform future development of child-focused diagnostic tools for paediatric PTB, I developed a conceptual framework to illustrate a) the requirements, and b) the relevance and impact of

improved microbiological diagnosis of PTB in children (Figure 1). Within this framework, I identified the following key considerations needing to be addressed for the improved diagnosis of PTB in children:

1. There is an immediate and unmet need to evaluate diagnostic strategies for PTB in children that are feasible and affordable for use in resource-limited settings, using the available resources and validated diagnostic tests (Figure 1; Block C). Consideration should also be given to the most appropriate placement of these strategies (e.g. primary care vs. hospital level), their function within the diagnostic framework (triage vs. replacement vs. confirmatory strategy) and diagnostic algorithms, and to the target patient population with relevant parameters such as age and severity of disease. 2. There is a long-term need to develop new diagnostic tests and strategies that

are specifically designed for children, with consideration of their unique and varied spectrum of disease and generally low mycobacterial concentrations in respiratory secretions (Figure 1; Block D).

3. Given the absence of an adequate diagnostic reference standard, the evaluation of new tests or diagnostic biomarkers for paediatric PTB will require well-designed and rigorously implemented studies. Key aspects of such studies would include detailed documentation of the study population and study sample, including eligibility/entry criteria, clinical evaluation and interpretation thereof, specimen collection procedures, including specimen storage and transport, and laboratory techniques. Relevant clinical co-variates should be included and described, including HIV status and immunological

markers, nutritional status, spectrum and severity of disease, and information about other possible clinical diagnoses/ co-morbidities. In addition, these well-characterized prospective paediatric cohorts should include a minimum duration of follow-up, well-defined disease phenotypes, and should include ill (symptomatic) controls. Longer term outcomes are needed to evaluate response to antituberculosis treatment, particularly in children with a clinical TB diagnosis (i.e. those not microbiologically confirmed), or clinical progress in the absence of antituberculosis treatment, in order to verify the initial diagnosis. Symptomatic children who are not initiated on antituberculosis treatment should be followed in the same way as children initially classified as TB cases, to evaluate (or validate) the performance of novel tests and clinical progress over time in this clinically relevant control group. Studies should be reported in sufficient detail and clarity as per Standards for Reporting of Diagnostic Accuracy (STARD) guidelines 62_{. Relevant paediatric}

populations with potential to gain from these diagnostic studies in the future should be included. Young children and infants should be specifically targeted for inclusion in such diagnostic studies, due to their disproportionate burden of TB-related morbidity and mortality, their vulnerability to rapidly progress to severe forms of TB, and the simultaneous diagnostic challenges, which are most prominent in the very young (Figure 1; Blocks A and B).

Improving the diagnosis of PTB in children (including earlier diagnosis and with microbiological confirmation) in clinical settings (Figure 1; Block E) will result in appropriate and more timely treatment of children (Figure 1; Block G), and may

prevent long-term morbidity and mortality, especially in children at the highest risk of TB and severe TB due to young age, HIV infection and malnutrition.

The improved diagnosis of PTB in children is also likely to lead to better reporting (notification) and better estimates of local-level and global burden of paediatric TB (Figure 1; Block F). Through continued research and advocacy, this could result in improved allocation of funding and resources to better prevent, diagnose and treat children from high TB-burden settings (Figure 1; Block G). Better diagnostic tools and biomarkers (Figure 1; Block D) will also allow more children to be included in interventional trials of novel antituberculosis drugs and improved (shorter, more potent, less toxic) regimens (Figure 1; Block I). The framework developed for characterising response to antituberculosis (and other) therapy (Figure 1; Block B) for the evaluation of diagnostic biomarkers which may be more sensitive than current diagnostic tests will in turn also provide an evidence base for evaluating antituberculosis treatment response, and therefore treatment efficacy, in children enrolled in such interventional trials (Figure 1; Block H). More effective, shorter and potentially less toxic treatment (Figure 1; Block J) will also result in better care for children with TB (Figure 1; Block G).

My doctoral research was therefore conceptualised and designed to address some of these aspects and research gaps, using the following methods:

1. A representative cohort of children with suspected PTB was enrolled and followed prospectively using rigorous and standard approaches for clinical evaluation, interpretation of chest radiography, specimen collection, processing and laboratory testing, and for reporting (Figure 1; Blocks A and

B). This cohort was designed to improve current diagnostic approaches for the bacteriological confirmation of PTB in children. In addition, the study also systematically followed the group of symptomatic controls (“non-TB cases”) as well as TB cases using identical methods, and documented clinical progress using standard approaches. I decided to focus on PTB (rather than extra- pulmonary TB - EPTB) because PTB is the most common form of TB in children, and while it is the most challenging to diagnose, especially in young children, it is also the form most likely to be confirmed microbiologically (with the exception of peripheral TB lymphadenitis - a relatively benign TB manifestation which is readily confirmed through microbiological testing from fine-needle aspiration biopsy; FNAB).

2. In this cohort, I used the available diagnostic platforms at the time, including liquid culture and the Xpert MTB/RIF assay (Xpert; Cepheid, Sunnyvale, CA, USA), to rigorously evaluate novel and potentially more feasible diagnostic strategies for use in resource-limited settings (Figure 1; Block C). Specifically, I evaluated:

a. The potential of stool as a child-friendly, readily available specimen to use for the diagnosis of children with suspected PTB, for detection of

M.tb using Xpert and culture.

b. Specimen pooling as a potentially more sensitive and cost-effective strategy using multiple respiratory specimens.

3. I evaluated the relationship between important clinical co-variates, frequently not well-characterized in many paediatric TB diagnostic studies, e.g. the

spectrum and severity of PTB disease, and the bacteriological yield of the different diagnostic approaches, including in specific groups of interest (e.g. HIV-infected children, young infants, those with higher bacillary burden), in order to make relevant, evidence-based recommendations on the utility of the various tested strategies for different clinical and epidemiological scenarios (Figure 1; Block A).

The scope of my doctoral research does not include the detailed analysis of long-term treatment response (Figure 1; Block F), or the development or evaluation of novel biomarkers (Figure 1; Block D). However, in addition to my research questions described above, I wanted to develop a rigorous scientific framework for future work in this field, and to establish relevant cohorts and methods for future work in this area (Figure 1; Blocks A and B). Novel diagnostic approaches which do not rely on the direct detection of the organism are clearly needed, but are beyond the scope of this doctoral dissertation. However, to address this additional important need, I followed all participants to 6 months regardless of treatment status, and established a well-characterised bio-repository of clinical specimens, including blood, urine and stool, for future evaluation of diagnostic and prognostic biomarkers for childhood TB.