Mini-symposium: Childhood TB in 2010
New specimens and laboratory diagnostics for childhood pulmonary TB: progress and prospects

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Summary

Childhood pulmonary TB (PTB) is under diagnosed, in part due to difficulties in obtaining microbiological confirmation. However, given the poor specificity of clinical diagnosis, microbiological confirmation and drug susceptibility testing is important in guiding appropriate therapy especially in the context of drug resistant TB. Confirmation is often possible, even in infants and young children, if adequate specimens are collected. Culture yield varies with the severity of illness, specimen type and culture method. Induced sputum is recognised as a safe procedure with a high diagnostic yield. Advances include optimised protocols for smear microscopy and modified culture techniques, such as the Microscopic Observation Drug Susceptibility Assay. Detection of Mycobacterium tuberculosis nucleic acid in respiratory specimens has high specificity but relatively poor sensitivity, particularly for smear negative disease. The recent development of an integrated specimen processing and real-time PCR testing platform for M. tuberculosis and rifampicin resistance is an important advance that requires evaluation in childhood TB.

Introduction

Globally, under diagnosis of childhood pulmonary tuberculosis (PTB) remains an obstacle to effective management. TB control programmes still focus predominantly on the diagnosis and cure of cases of smear-positive adult TB, as these are the major drivers of TB transmission. Whilst adult TB cases are often easily recognizable, due to typical radiological features and a positive sputum smear, childhood TB is frequently more difficult to diagnose. The clinical and radiological features of childhood TB are often non-specific and subject to variable interpretation.1 Structured diagnostic scoring systems based on clinical and radiological findings and tuberculin skin testing (TST) show high variability in case yield and very poor agreement.2

The diagnosis is even more problematic in HIV-infected children, since clinical and radiological features overlap with other infections and anergy to the TST is common.3 Clinical scoring systems have not been adequately evaluated in HIV-infected children, but it is likely that their performance will be even poorer in this patient population.

Microbiologic confirmation of PTB is still rarely attempted in children, especially in primary care settings, in contrast to adults where this is the accepted standard of care. This is due to the incorrect perception that respiratory specimens are difficult or impossible to obtain in children, the lack of infrastructure or trained staff to obtain such specimens and the lack of policy regarding microbiologic confirmation in children. However, even when samples can be obtained, since disease is typically paucibacillary, the yield of direct acid-fast smear microscopy is very low4 and prolonged mycobacterial culture is required. As a result, microbiological confirmation may be delayed by weeks. This has important implications for a disease that may progress rapidly in young children, with associated morbidity and mortality. Extra-pulmonary TB is common in young children and poses particular challenges for specimen collection and culture.

The major advantages of obtaining microbiological confirmation are the ability to make a definitive diagnosis and to perform drug susceptibility testing to exclude drug-resistant TB. In the era of increasing multidrug resistant (MDR) and extensively drug resistant (XDR) TB this information becomes critical in order to guide appropriate therapy.

However, mycobacterial culture is frequently negative in children with clinically diagnosed PTB, particularly amongst less ill patients in a primary care setting.5 This may represent the poor specificity of clinical diagnosis or alternatively, the impaired sensitivity of culture for childhood TB. This presents a fundamental problem in assessing the performance of any novel diagnostic test or clinical algorithm as there is no reference standard which is both highly sensitive and specific to which results can be compared. A further implication is that clinicians are often wary to discontinue TB therapy when a negative culture result is obtained.

This review will address the progress made in recent years in obtaining laboratory confirmation of PTB in children. The diagnosis of latent TB infection and the use of serological tests and interferon-gamma release assays are considered elsewhere in this edition.

Section snippets

Obtaining representative specimens from the lower respiratory tract of children

Microbiological confirmation of TB in young children is not routinely attempted in many high burden settings due to the difficulty in obtaining samples and the poor performance of smear microscopy. However, if facilities for mycobacterial culture and drug susceptibility testing are available, such confirmation is invaluable.

Since young children are frequently unable to expectorate, additional procedures are often required to obtain samples from the lower respiratory tract. For many years the

Improvements in smear microscopy(Level A)

There have been incremental advances in the performance of smear microscopy for the rapid detection of MTB. It is clear that concentration of specimens by centrifugation18 and the use of fluorescent microscopy19 increase the yield of smear microscopy [Table 1]. Until recently, one of the drawbacks of fluorescent microscopy has been the need for a costly, short-lived mercury vapour light source. The recent availability of low cost, long-lived light emitting diode (LED) light sources for

Improvements in culture techniques

There is no doubt that liquid culture systems with continuous monitoring for mycobacterial growth (such as MB/BacT [Biomerieux, Marcy l’Etoile, France], BACTEC 9000 [Becton Dickinson, NJ, USA] and the mycobacterial growth indicator tube [MGIT; Becton Dickinson]) are a significant advance over solid culture (typically Löwenstein Jensen, LJ) [Table 1]. In adult studies, mean time to detection is substantially lower for automated liquid culture (13.2 vs. 25.8 days for MGIT vs. LJ in a recent

Detection of M. tuberculosis nucleic acid

Whilst detection of live MTB is likely to remain the gold-standard for diagnosis in the immediate future, nucleic acid amplification assays (NAA) for the detection of MTB DNA or RNA are finally coming of age. There are obvious attractions to NAA. These assays are theoretically highly sensitive, able to detect very low copy numbers of nucleic acid, rapid (results typically available on the same day), may not require biosafety level 3 facilities and hence may be deployed closer to the point of

Other tests

There are two novel diagnostic modalities which have not, to our knowledge, been evaluated in children, but which are likely to be tested in this population in the near future; the urinary lipoarabinomannan (LAM) assay and tests for volatile organic compounds in the breath. The LAM assay is an ELISA-based test for a mycobacterial glycolipid. The sensitivity of urine LAM testing for adult disease has varied widely (44%-67%)38, 39 with the higher estimates in HIV co-infected patients with

Conclusion

Advances in the diagnosis of childhood TB in the past decade have included the identification of alternative specimen types such as induced sputum and nasopharyngeal aspirate as well as improvements in smear microscopy and liquid culture systems. A number of novel and exciting candidates have been identified for diagnosis of adult TB, such as integrated real-time PCR detection systems, urine LAM and testing for volatile organic compounds in breath. There is a clear need for a large, prospective

Funding sources

Mark Nicol is funded for TB research by the Wellcome Trust, EDCTP, NIH, the NHLS research Trust and South African MRC. The funding sources did not play any role in the writing of this manuscript.

Heather Zar is funded for TB research by the NIH, USA, EDCTP, MRC, South Africa and National Research Foundation, South Africa. The funding sources did not play any role in the writing of this manuscript.

Educational Aims

  • To describe current state-of-the-art for diagnosis of pulmonary tuberculosis in children

  • To discuss the advantages and limitations of various specimen types for diagnosis of pulmonary tuberculosis in children

  • To discuss the role and limitations of nucleic acid amplification assays in the diagnosis of pulmonary tuberculosis in children

Practice Points

For diagnosis of pulmonary tuberculosis in children:

  • Microbiological confirmation should be sought

  • Sputum induction is a safe procedure with a high diagnostic yield

  • Samples should be processed by chemical lysis of sputum, centrifugation, fluorescent microscopy and liquid culture

  • The results of ‘in-house’ nucleic acid amplification assays should be interpreted with caution and such assays require rigorous clinical evaluation prior to use

Research Directions

  • The performance of novel integrated nucleic acid amplification systems (such as GeneXpert) for the diagnosis of pulmonary tuberculosis in children

  • The utility of nasopharyngeal aspiration for the diagnosis of pulmonary tuberculosis in children

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