Microbiological diagnostic procedures in respiratory infections: mycobacterial infections

PAEDIATRIC RESPIRATORY REVIEWS (2007) 8, 221–230

MINI-SYMPOSIUM: MICROBIOLOGICAL DIAGNOSTIC PROCEDURES IN RESPIRATORY INFECTIONS

Microbiological diagnostic procedures in respiratory infections: mycobacterial infections David Andresen Department of Microbiology, and Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, Australia KEYWORDS mycobacterium; tuberculosis; diagnostic tests; tuberculin skin test; gamma-interferon; culture; microscopy; nucleic acid detection

Summary This article will review traditional and newer microbiological techniques for the diagnosis of mycobacterial respiratory infections. It will concentrate on the diagnosis of infections due to Mycobacterium tuberculosis, the main mycobacterium causing respiratory infections of clinical and public health importance. The diagnosis of respiratory disease associated with non-tuberculous mycobacteria (NTM), particularly in children with underlying airway pathology such as cystic fibrosis (CF) or bronchiectasis, will be briefly discussed. With respect to the diagnosis of tuberculosis (TB), the review will concentrate on the diagnosis of patients with symptoms and/or signs of clinical disease, rather than the detection of exposure or asymptomatic infection. It will not specifically address the assessment of pre-test probability based on clinical or epidemiological factors, the use of radiological investigations or the investigation of extrathoracic lymph node disease or chest wall disease. The role of newer diagnostic modalities including nucleic acid detection (NAD) and gamma-interferon assays in paediatric practice will be reviewed, and suggestions made as to how they may fit into contemporary diagnostic algorithms. ß 2007 Elsevier Ltd. All rights reserved.

CLINICAL SYNDROMES Mycobacterium tuberculosis may affect any part of the respiratory tract. The presence or absence of communication with the airway is important both for diagnostics (the ability to detect the organism in airway or gastric specimens) and for transmissibility. ‘Communicating’ respiratory tuberculosis (TB) may affect the mouth, tonsils, epiglottis, larynx, trachea, bronchi or distal pulmonary parenchyma. ‘Non-communicating’ respiratory sites include the intrathoracic lymph nodes (although rupture into adjacent airways may occur), other mediastinal structures, pleural and pericardial surfaces, and the chest wall, particularly ribs, joints and vertebrae. E-mail address: [email protected] 1526-0542/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2007.07.002

The non-tuberculous mycobacteria (NTM) generally affect the lymph nodes, airways or lung parenchyma. Their clinical significance in respiratory specimens may be difficult to ascertain, but the Mycobacterium avium complex and the rapidly growing mycobacteria (particularly Mycobacterium abscessus) have been associated with more rapid progression of lung disease in cystic fibrosis,1 and with pulmonary parenchymal or endobronchial infections, particularly (but not exclusively) in immunosuppressed hosts.

AGE GROUPS Infants under 12 months of age Most infants with active TB are symptomatic, and an infectious adult contact is often identifiable. Seventy per cent of

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KEY POINTS  Several laboratory tests may be useful to confirm or disprove the diagnosis of mycobacterial respiratory infections in children.  The diagnostic yield from culture is lower in children than adults, but the isolation of Mycobacterium tuberculosis strongly supports a diagnosis of TB and allows susceptibility testing. Novel culture methods appear promising.  The greater the number of specimens and specimen types the greater will be the diagnostic yield, but with diminishing incremental returns from each additional specimen.  Nucleic acid detection assays have been inadequately validated in children, and their specificity may be suboptimal. They may have a role in selected difficult diagnostic situations.  The tuberculin skin test is a widely available test for TB infection, but a negative result cannot exclude a diagnosis of tuberculosis.  In vitro gamma-interferon assays have also been inadequately evaluated in children. Their main role may be in patients with a positive tuberculin skin test but past BCG vaccination, to help define whether the BCG or TB infection is responsible for the TST reactivity.  Criteria exist to help ascertain the significance of non-tuberculous mycobacteria isolated from children with underlying airways disease.

infants with active TB have respiratory infection, often mediastinal nodal or miliary disease. Mediastinal lymphadenopathy may compress large airways,2 and lung parenchymal cavitation is rare.3,4 Hepatosplenomegaly is common, but the tuberculin skin test (TST) is usually unreactive.2 The bacillary load is usually higher than in older children, and three gastric aspirates yield a definite diagnosis by microscopy or culture in >70% of infants with pulmonary infection.2,5

Children Most children who develop symptomatic TB do so within 6– 12 months of infection, and active disease is most common under 5 years of age.6 Smear-positive disease is less common than in infants or adults,7 and a definitive diagnosis is more difficult to achieve. Presumptive diagnoses are frequently made, and treatment commenced, on the basis of exposure to an infectious adult, a positive TST, and either a typical clinical syndrome or an abnormal chest radiograph (CXR).6,8

Adolescents Adolescents are more likely than younger children to be symptomatic, to have cavitating pulmonary disease, and to

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be smear- and culture-positive on sputa or gastric aspirates. While these features are more ‘adult like’, they still have a relatively high rate of extrapulmonary disease.9

DETECTION OF M. TUBERCULOSIS Specimen types Sputum Young children will rarely be capable of producing an adequate sputum specimen and even older children and adolescents may have difficulty. Methods for the collection and transport of sputum specimens have been well described elsewhere.10

Gastric aspiration Aspiration of about 50 ml of gastric contents should be performed fasting, ideally while still recumbent after overnight sleep.10 For this reason it is usually performed in hospitalized patients. It has been suggested that strict adherence to the recommended collection techniques improves the yield of the procedure,11 as does the collection of specimens on three consecutive days.

Induced sputum The use of nebulized hypertonic saline to induce coughing allows the production of respiratory specimens in children who cannot expectorate voluntarily.12 The yield from a single induced sputum specimen is similar to that of three daily gastric aspirates, and is increased further by repeated procedures. The technique has been shown to be well tolerated, safe and effective even in very young children and older infants.13 The main objections to sputum induction are that children who are severely hypoxic prior to the procedure may be at risk of respiratory decompensation, and that transmission to healthcare workers is possible without careful environmental control and use of personal protective equipment. Detailed methods for the collection and transport of induced sputum specimens have been well described elsewhere.10

Bronchoscopy The diagnostic role of bronchoscopy has been controversial, and bronchial washings have been inferior to three gastric aspirates in terms of recovery of mycobacteria by culture.14–16 The procedure may however allow the isolation of M. tuberculosis in some patients in whom three gastric lavage specimens are negative.17 Apart from its sampling ability, bronchoscopy may assist with the diagnosis of airway sites of TB infection and help assess the presence and severity of extrinsic airway compression by intrathoracic lymph nodes. At this stage the decision of how widely to use bronchoscopy is probably a local one, depending on

MYCOBACTERIAL INFECTIONS availability, expertise and enthusiasm of the operators.18 If sputum if produced post-bronchoscopy it should also be submitted for microscopy and culture.10

Pleural fluid or biopsy Children with tuberculous pleural effusions or empyema may also have pulmonary parenchymal involvement, in which case the causative organism may be recovered from respiratory tract specimens or gastric aspirates. Across all age groups the diagnostic yield from pleural aspiration is only 20% by microscopy,19 and poor even by culture. The detection of acid-fast bacilli (AFB) is substantially improved if pleural biopsy is performed, and the histological observation of typical granulomata may allow the commencement of therapy pending culture confirmation.10 Amongst 39 children with tuberculous pleural effusion20 the diagnostic yield was 5.5% by AFB microscopy and 44% by pleural fluid culture. In contrast, pleural tissue specimens were diagnostic by culture or histology in almost 80% of patients in whom biopsy was performed. The use of newer diagnostic techniques for tuberculous pleural infections is discussed in subsequent sections.

223 techniques25 may improve this substantially. AFB microscopy has been shown to have sensitivity below 20% on gastric aspirate or sputum specimens from children.26–28 As a result, a negative microscopy result cannot exclude TB, but a positive result may be very useful in patients with a clinically compatible illness. Positive microscopy may be non-specific due to the presence of NTM in patients with underlying structural lung disease such as cystic fibrosis or bronchiectasis. In these patients nucleic acid detection (NAD) assays for M. tuberculosis may have an important role pending culture and identification results. Traditional acid-fast staining techniques include the Ziehl–Neelsen (hot) and Kinyoun (cold) methods.10,29 Fluorescent microscopy using Auramine O with or without Rhodamine B allows the scanning of slides at lower magnification and therefore may increase the sensitivity of microscopy while reducing the operator time required.30 There may be some marginal reduction in specificity compared with traditional Ziehl–Neelsen and Kinyoun stains,30,31 but a traditional stain may be performed ‘over’ the fluorescent stain on the same slide if confirmation is required and resources allow.29

AFB culture Tissue biopsy Formal tissue biopsy may be important to exclude alternative diagnoses, particularly in children with mediastinal or intrathoracic nodal masses when less invasive tests have not been successful. It is rarely required for pulmonary parenchymal TB. If tissue specimens are obtained, then examination by histological techniques as well as smear and culture is recommended.

Specimen processing Traditional specimen processing techniques for M. tuberculosis microscopy and culture have been summarized elsewhere.10 The use of a mucolytic agent such as Nacetyl-l-cysteine and a decontaminant such as sodium hydroxide is standard practice for respiratory and gastric specimens, but is not required for sterile site specimens such as pleural fluid. Microscopy of unprocessed sputum smears is sometimes performed in sites with limited resources. Concentration by centrifugation improves sensitivity of both microscopy21 and culture. Novel processing regimens22,23 have been suggested to improve the yield of smear and/or culture; however, processing methods which allow greater smear sensitivity generally do so at some cost to specificity.

AFB microscopy Traditional specimen processing and centrifugation techniques yield a lower limit of detection by microscopy of about 5000 organisms/ml,24 although novel processing

Culture is the most sensitive and specific means of detection of Mycobacterium tuberculosis in body fluids, with an estimated lower limit of detection of between 10 and 100 organisms per mL. As well as its sensitivity, culture of the organism allows drug susceptibility testing and is therefore most critical where drug resistance is of concern for epidemiological reasons.6 Unfortunately the diagnostic yield from culture rarely exceeds 50% in children, even in sophisticated labs when multiple specimens are submitted.28,32,33 Children are less likely to produce sputum, so alternative specimen collection techniques are usually required. In the USA between 1985 and 1998 only 28% of children diagnosed with TB had positive culture results.34 Culture of three consecutive gastric aspirates can achieve a sensitivity of 70% in infants and 30–50% in children.5,32 The incremental yield from culturing bronchoscopic lavage (BAL) fluid or induced sputum in a child who has had three gastric aspirates has not been well defined. Blood cultures are rarely of diagnostic value except possibly in immunosuppressed hosts.35 Mycobacterial culture may be performed using solid media such as the egg-based Lowenstein–Jensen slopes or the Middlebrook series of agars, or using liquid broths. A novel culture method [microscopic observation drug susceptibility (MODS)] using a broth culture medium in 24well plates has recently been designed for use in resourcepoor settings. As well as being substantially cheaper than the use of commercial broth culture media, it incorporates drug susceptibility testing in the initial culture phase, allowing rapid detection of drug resistant isolates. It has been shown to have superior sensitivity to solid (Lowen-

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stein Jensen) culture media with a substantially reduced (median 7 day) time to positivity.36 It has also been shown to be superior36 or equivalent37 to commercial broth culture systems in terms of yield and time to positivity. In a limited paediatric evaluation,38 MODS had a significantly higher culture yield than Lowenstein–Jensen slopes, particularly in smear-negative specimens, with reduced time to positivity. This methodology appears to hold great promise for settings where culture has not previously been attempted, where commercial broth culture methods are prohibitively expensive or where multidrug-resistant TB is common.39 Identification and susceptibility testing techniques for mycobacteria isolated in culture have been well described elsewhere.10 DNA hybridization probes have become widely used rapidly to distinguish M. tuberculosis from the NTM in positive cultures. A commercial line probe hybridization assay can identify mutations associated with rifampicin resistance, and has excellent performance when applied to positive cultures.40 It can also be applied directly to smear-positive clinical specimens, although the sensitivity is lower in this setting. If available, its use is recommended when resistance is suspected on epidemiological or clinical grounds.

Nucleic acid detection (NAD) assays The detection of specific mycobacterial DNA sequences by NAD techniques, such as PCR, has the potential to be a useful and rapid diagnostic modality. In smear-positive specimens, a TB NAD assay may allow confirmation of the species of mycobacterium being observed, and in this context an approximate sensitivity of 95% and specificity of 98% can be expected.10 In smear-negative specimens, a sensitive NAD assay might allow the diagnosis of TB to be made more rapidly than by culture. However, the evaluation of NAD assays for TB diagnosis has been hampered by several methodological problems related to the evaluation of NAD-positive but culture-negative specimens. NAD assays do have the potential for false positive results due to contamination or cross-reactivity, but may also detect the presence of DNA from nonviable organisms. As NAD assays have a similar sensitivity to culture in smear-negative specimens,41 there will inevitably be a proportion of NADpositive, culture-negative patients and specimens. Unless other specimens from the same patient are culture positive, whether these ‘additional’ positive NAD results represent false or true positives is probably best assessed by careful longitudinal clinical evaluation, including response to any therapy given.42 NAD assays may be ‘inhouse’ (designed by the lab performing the assay) or commercial. Meta-analyses of NAD assays41,43 applied to sputum specimens from predominantly adult populations have identified too much heterogeneity to derive meaningful summary measures of test accuracy. The use of nested (two-step) amplification

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protocols or the multicopy gene target IS6110 were associated with improved test performance,43 and the use of the commercial Amplicor (Roche) TB PCR test was generally associated with lower sensitivity but better specificity than inhouse tests in studies with predominantly adult populations.41 In an early paediatric study, an inhouse IS6110 assay correlated well with the presence of clinically active disease as defined by American Thoracic Society (ATS) criteria.44 It was, however, positive in many patients with negative culture results, particularly those defined on clinical grounds as having TB infection without disease. This finding of positive gastric aspirate NAD results in culture-negative children who clinically have TB infection without disease has been noted by other groups.45,46 The best evaluations of NAD tests for the diagnosis of respiratory tract TB in children have been reported by Gomez-Pastrana et al.47,48 They used an inhouse nested PCR, re-tested all PCR-positive specimens, and applied a multifactorial reference standard combining chest computed tomography (CT) scanning, longitudinal clinical evaluation and culture of all specimens. Specimens were mostly gastric aspirates, with a small number of bronchial lavages. In their first study, AFB smear and culture had sensitivities of 13.5% and 37.8%, respectively, both having 100% specificity.47 Inhouse PCR had a sensitivity of 56.8% and a specificity of 93.8%. All ‘false positive’ PCR results, however, occurred in children clinically assessed as having infection without disease, but who had mediastinal lymphadenopathy on CT scanning. They subsequently compared their inhouse assay to the Roche Amplicor M. tuberculosis PCR assay in a similar study design on an apparently overlapping patient population.48 This demonstrated a sensitivity of 60% and specificity of 97% for the inhouse assay, which was superior to the sensitivity of 44% and specificity of 94% for the Amplicor PCR. Other groups have also noted poor specificity results of NAD tests in children. Specificities of only 95% for a single-round and 88% for a nested PCR assay were observed on mixed gastric and respiratory specimens46 and only 80% specificity was obtained with a singleround assay on gastric specimens.33 In gastric specimens there may be a high frequency of discordance between NAD and culture results.49 The role of TB NAD tests on pleural fluid has also been controversial. While inhouse assays vary widely in their performance characteristics, studies in predominantly adult populations have shown that commercial TB NAD assays have high specificity (>95%) but widely variable sensitivity.50 There have been few evaluations of TB NAD applied to pleural fluid in children. Mishra et al evaluated an inhouse IS6110 PCR on paediatric pleural and pericardial effusions and demonstrated a specificity of only 90%, which is probably too low to be helpful outside a high prevalence environment.51 In summary, there are insufficient data to recommend the routine use of NAD assays such as PCR for the diagnosis of

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paediatric TB. NAD assays are not sufficiently sensitive to exclude TB if they are negative. They are also not sufficiently specific for a positive result confidently to establish a diagnosis of TB when the pre-test suspicion of disease is low. A positive result from a commercial or well-validated inhouse (particularly nested or IS6110) assay may have a role in difficult diagnostic situations, where the pre-test likelihood is intermediate and a rapid diagnosis is required. Due to the technical requirements and cost of NAD assays, they may not be suitable in some resource-poor settings. Although there have been no good clinical evaluations in immunosuppressed children, NAD assays may ultimately have a more important role in their evaluation since the TST and gamma-interferon assays may be negative, the ‘organism load’ may be higher and non-tuberculous mycobacteria may lead to misleading positive AFB smear results.

DETECTION OF THE HOST RESPONSE TO M. TUBERCULOSIS Tuberculin skin test The TST involves the assessment of cell-mediated immunity to antigens of M. tuberculosis, exhibited by the development of a delayed type hypersensitivity reaction assessed at 48 72 h after single (Mantoux) or multiple (Heaf, Tine) intradermal injections. Tuberculin reactivity develops between 3 weeks and 3 months after exposure to M. tuberculosis.24 Multiple puncture TSTs are no longer widely used. The use of the Heaf test was discontinued in Britain in 2005 due to issues of equipment supply, and its use has been discouraged in the USA for many years.10 The Mantoux test is now the main TST in use worldwide, although different antigen preparations such as RT 23 and PPD are available and may have slightly different performance characteristics.52 Since the TST assesses T-cell reactivity to mycobacterial antigens, it is unable to distinguish between TB ‘infection without disease’ and clinically active tuberculous infection. It is of great value in assessing exposure to TB and targeting preventative chemotherapy, such as isoniazid, in individuals without clinical disease. It also has an important role in assessing patients with active clinical disease which may be due to TB, but some caveats apply and these are discussed below. The diameter of induration which defines Mantoux positivity may vary depending on the clinical setting in which the test is used.10 The selection of a cut-off of 5, 10 or 15 mm represents a trade-off of sensitivity (maximized by a low cutoff) against specificity (maximized by a high cut-off) along a receiver operator characteristic curve. In situations where the pre-test probability of TB is high, such as a child with an abnormal chest radiograph who has been in contact with an adult with cavitatory TB, the ATS recommends a cut-off of 5 mm induration in order to maximize sensitivity. In a screening setting, where the pre-test probability of TB is low, a cut-off of 15 mm induration is used in order to

225 maximize specificity.10 Guidelines produced by other bodies such as the World Health Organization (WHO) do not attempt to manipulate cut-off values, and instead use a single threshold for positivity which depends on the individual’s Bacille Calmette-Guerin (BCG) vaccination status.24 The sensitivity of TST for the diagnosis of active TB in children probably does not exceed 90%.27,53 This sensitivity may be substantially lower in infants, HIV positive or otherwise immunocompromised children, malnutrition and extensive or miliary TB.32,53 Some immunocompetent children with initially negative TST’s develop skin test reactivity after successful antituberculous chemotherapy, but a proportion remain persistently nonreactive.53 As a result of these characteristics, a negative TST cannot reliably exclude active tuberculous disease, particularly in infants or in children with any of these associated conditions. False positive Mantoux tests, particularly using the lower threshold of 5 mm induration, may be associated with past vaccination with BCG or with exposure to non-tuberculous (environmental) mycobacteria, which share antigens with M. tuberculosis. The specificity of the test for TB infection (with or without active disease) is >95% in a developed world setting where BCG use is uncommon.54 The degree to which TST interpretation is confounded by past BCG vaccination is somewhat controversial. Most (80 90%) children will have a negative TST 5 years after infant BCG vaccination55 and an induration diameter >15 mm is unlikely to be due to BCG alone.52 In summary, TSTs such as the Mantoux test represent useful and readily available adjunctive tests in the assessment of children with suspected active TB but are insufficiently sensitive reliably to exclude the diagnosis if they are negative. Positive results cannot distinguish between past TB infection and active TB disease, and in children with <15 mm induration the BCG vaccination history needs to be taken into consideration. In the appropriate clinical context, after exclusion of alternative diagnoses, a positive TST result may support a decision to commence antituberculous chemotherapy. Attempts to culture the organism are still recommended, particularly if drug resistant TB is suspected on epidemiological or clinical grounds.

Tests of humoral immunity The ability to detect antibodies specific to M. tuberculosis has been investigated by many investigators with a view to developing a serological test for TB infection. Several commercial assays have demonstrated disappointing performance in predominantly adult populations. In one casecontrol evaluation of seven serodiagnostic tests in adults,56 the best performing test had a sensitivity of 55% and a specificity of 90%. These performance characteristics are substantially poorer than those expected from the TST, and did not support the routine use of humoral serodiagnosis.

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Prior to this decade, the most favourable paediatric results were obtained in an evaluation of an Antigen 60 IgG ELISA assay. In a BGC vaccinated population, the use of age-specific cut-off values allowed a sensitivity of 68% for active TB disease with a specificity of 98%.57 However, subsequent paediatric studies showed unacceptably poor performance with this and other antigens,58–60 and research attention shifted to the in vitro gamma-interferon (T-cell activation) assays discussed below. Recently, an Indian group published promising results using antigens of the excretory–secretory system of M. tuberculosis such as ES-31.61,62 If these results are replicated and extended, and an assay is commercialized, humoral approaches may yet find a role in the diagnosis of paediatric TB.

In vitro gamma-interferon tests Two commercial tests based on the measurement of gamma-interferon production after the in vitro activation of T-cells by exposure to antigens of M. tuberculosis have recently become available. The QuantiFERON-TB Gold ELISA (Cellestis, Victoria, Australia) and the T-SPOT TB (ELISPOT) assay (Oxford Immunotec, Abingdon, UK) employ the ESAT-6 and CFP-10 antigens, which are lacking in BCG and common environmental mycobacteria such as M. avium. They may therefore be more specific than the TST, or less influenced by BCG vaccination status. As with the TST they are unlikely to be able to distinguish active TB disease from TB exposure. The in vitro T-cell activation assays may also have operational advantages in that the return of the patient for ‘reading’ at 48–72 h is not required, and boosting by repeated testing cannot occur. The personnel and skills required to perform these tests also differ substantially from the TST. The United States Centers for Disease Control (CDC) has recommended that the QuantiFERON-TB Gold may be used ‘‘in all circumstances in which the TST is currently used’’.63 In predominantly adult evaluations of these assays,64 the sensitivity for active TB varied from 80% to 100%, and in several studies appeared to be lower than that of the TST. The specificity of gamma-interferon tests also appeared, unexpectedly, to be modestly reduced by prior BCG vaccination in some evaluations. In a prospective study of adults and children with suspected TB, positive results were obtained in 14 of 21 patients with active TB who had a Mantoux test, 17 of 24 who had a QuantiFERON-TB Gold, and 20 of 24 who had a T-SPOT TB test.65 Indeterminate results were more often found with the QuantiFERON-TB Gold, particularly in children under 5 years of age. Immunosuppressed patients were more likely to have indeterminate results in both gamma-interferon assays. There have been few methodologically sound evaluations of commercial gamma-interferon assays in children. Responses to ESAT-6 or CFP-10 were found in only 70% of

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children with active TB in one study, and were more common in culture-positive disease.66 In a study of TBexposed 11–15 year old British schoolchildren, gammainterferon tests correlated better with degree of exposure to the index case than the TST, and were less likely to be confounded by past BCG vaccination.67 However, in a similar study from The Gambia, TST positive/ELISPOT negative children were increasingly common with higher levels of exposure to smear-positive TB cases, and BCG vaccination status did not affect the performance of either assay.68 As with the evaluation of NAD assays for the diagnosis of TB, the lack of a satisfactory simple gold standard for comparison makes the formal evaluation of the performance of the gamma-interferon tests difficult. Ideally, a multifactorial reference standard utilizing conventional diagnostic modalities, observation over time and response to therapy should probably be used. In a well-performed prospective evaluation of an inhouse ELISPOT assay in an area of high HIV prevalence,69 the ELISPOT had significantly better sensitivity (83%) for proven or highly probable tuberculous disease than a Mantoux reaction of 5 mm or greater (66%). The superiority of the ELISPOT test was more marked in very young, malnourished or HIV positive children. However, 30% of TST-negative children in whom active TB was clinically excluded also had a positive ELISPOT assay. In a high-risk Australian population, 70% of children with latent TB infection as defined by a positive TST had a negative QuantiFERON-TB Gold test, and 17% of QuantiFERON-TB Gold assays gave indeterminate results.70 The TST-positive/QuantiFERON-TB Gold negative results did not appear to be explained by past BCG vaccination. All nine children with active TB disease had a positive QuantiFERON-TB Gold result. Despite the CDC endorsement of the QuantiFERONTB Gold test, there are insufficient data regarding its performance in children to support its routine use in place of the TST. Unless the operational advantages are compelling (such as children unlikely to return for TST reading) there are sufficient concerns regarding the rates of indeterminate results, and possibly lower sensitivity than the TST,71 to caution against such a direct substitution. As with the TST, the gamma-interferon assays are not sufficiently sensitive confidently to exclude active TB on the basis of a negative result. Until better paediatric evaluations are available, the gamma-interferon tests may be of some use in clarifying the basis of a positive TST result in BCG-vaccinated children. They may also have a role in confirming a negative TST result in patients who may not be capable of a skin test response, such as the very young, the immunosuppressed, and the malnourished or debilitated. While a positive gamma-interferon test result in these children would suggest TB exposure, it is important to emphasize that a negative result cannot exclude TB infection or disease.

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OTHER TESTS

Lung parenchymal disease

Measurement of adenosine deaminase (ADA) levels has been proposed as a useful diagnostic adjunct for tuberculous pleural effusions. A recent exhaustive assessment across all age groups concluded that ADA assays on pleural fluid have reasonable diagnostic performance, although the methodological quality of the primary studies was less than ideal.72 A recent small study of children with effusions which were predominantly pleural showed that ADA had similar performance to PCR on pleural fluid, with a sensitivity of 81% and a specificity of 75% for proven or probable tuberculous disease.73 The authors also analysed the combination of ADA >38 IU/L and pleural fluid lymphocyte count >275/mm3, and showed that either test being positive had a sensitivity of 100%, while the combination of both tests being positive conferred a specificity of 92%. ADA in pleural fluid may be of some diagnostic value, if locally available, but its role in a paediatric setting is not well defined at present. The diagnostic value of lysozyme levels in pleural fluid or of the fluid-to-serum lysozyme ratio has also been explored. In predominantly adult evaluations, the best performance was obtained using the pleural fluid-to-serum lysozyme ratio, which was capable of sensitivity and specificity results of >90%.72 However, no paediatric study has reproduced these results. A small study of children with effusions which were predominantly pleural showed that ‘raw’ fluid lysozyme levels were superior to the fluid-toserum lysozyme ratio.73 Purulent non-tuberculous effusions also had elevated lysozyme levels, but if these were excluded then the diagnostic performance of this assay was excellent. Until better paediatric evaluations are available, the routine use of pleural fluid lysozyme levels is not recommended. While antigen detection techniques have been proposed for the diagnosis of TB in adults, no such assays have become widely available or been adequately evaluated in paediatric populations.

When laboratory resources are of a reasonable standard, most patients with disease at a ‘communicating’ respiratory site should have an attempt at detection of the causative organism by microscopy and culture (using whichever laboratory methods are locally available) of three sputa (in older children who can expectorate), three early morning gastric aspirates, or an induced sputum specimen in children over 6 months of age. BAL fluid may also be used if bronchoscopy facilities and expertise are locally available, particularly in immunosuppressed children where other opportunistic infections require exclusion. BAL fluid should probably not be relied upon as the only specimen type for the isolation of M. tuberculosis in children. The decision whether to use one or other (or several) of these techniques will depend on local expertise, enthusiasm and facilities. In general, the greater the number of specimens and specimen types, the higher will be the yield, but with diminishing incremental returns for each additional specimen. In children with a compatible clinical illness, a positive AFB microscopy result will usually be sufficient evidence to justify the commencement of antituberculous chemotherapy. In smear-positive children with underlying lung disease, a TB NAD assay may be useful to clarify whether the observed bacilli represent M. tuberculosis or NTM. Even with multiple specimens the sensitivity of microscopy is low, so in smear-negative children additional diagnostic tests may be employed while awaiting culture results. A strongly positive TST may provide useful presumptive evidence of TB exposure, though patients with a weaker positive result and a history of BCG vaccination may benefit from an in vitro gamma-interferon assay if this is locally available. On the other hand, a negative TST and/or gamma-interferon assay cannot exclude the diagnosis of active TB. In patients in whom the possibility of TB remains intermediate after the above investigations, or in immunosuppressed children, if a commercial or well-validated inhouse NAD assay is available then positive results may be of some diagnostic help despite the suboptimal specificity of some TB NAD assays in children. In patients who are started on antituberculous chemotherapy on the basis of high clinical suspicion, possibly supported by positive TST, gamma-interferon assay or NAD results, and in whom TB cultures are ultimately negative, it should be remembered that the yield from culture is only about 50% at best. If these children are tolerating their therapy, their respiratory disease is improving and no alternative diagnosis has become apparent, then it will usually be appropriate to complete their course of TB therapy.

DIAGNOSTIC ALGORITHM In patients with respiratory disease in whom TB is suspected, the likelihood of this diagnosis is initially assessed implicitly or explicitly74,75 on the basis of epidemiological, clinical and radiological features. None of these features is usually sufficiently sensitive or specific to allow the confident diagnosis or exclusion of TB, so adjunctive investigations are generally required. In high-prevalence settings where laboratory resources are scarce or basic, many children with respiratory disease typical of TB who have been recently exposed to a smear-positive contact will be treated for TB without further investigations. This is not recommended if drug-resistant TB is prevalent and the susceptibility results from the index patient are not known.

Pleural effusion or thoracic empyema Pleural TB is generally a paucibacillary process, and the yield from fluid microscopy is low. The TST will nearly always be

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positive in immunocompetent children, so a negative result is strong evidence against the diagnosis. The diagnostic yield will be maximized by the examination of pleural biopsy specimens as well as pleural fluid. Biopsy is particularly recommended if the diagnosis remains in doubt after less invasive testing or in settings where drug-resistant TB is common. In non-purulent effusions the use of ADA, possibly in combination with absolute fluid lymphocyte count, may be used if locally available, although the paediatric evaluation of these tests has been limited. It is unclear whether the improved sensitivity of NAD assays compared with microscopy19 substantially aids diagnosis, given that the specificity of many NAD tests remains suboptimal. In settings where the pre-test probability of TB is moderately high and pleural biopsy cannot be performed for technical reasons, NAD assays on pleural fluid may have a role. From limited data, NAD assays may not perform much better than pleural fluid ADA levels, so ADA may be a satisfactory alternative in settings where NAD tests are prohibitively expensive or complex.

Diagnosis of TB in HIV infected children The diagnosis of TB in HIV infected children is complicated by the reduced sensitivity of immunological diagnostic techniques such as the TST and gamma-interferon assays. There is also greater clinical need rapidly to make a firm diagnosis, and a wider range of alternative pathological processes to consider. The use of a 5 mm cut-off for a positive Mantoux test is recommended. Gamma-interferon assays may be preferable to the TST in this population,69 but a negative test cannot exclude tuberculous disease. On biological grounds it would be reasonable to assume that smear, culture and possibly NAD assays would have a greater diagnostic role in HIV infected children due to an increased bacillary load. However, yields from smear and culture have not always been higher in HIV positive children.76 Also, firm data regarding the performance of NAD assays in this setting are lacking. Positive NAD assays may lend support to the diagnosis, particularly the commercial assays which have better specificity. If the diagnosis remains unclear despite ‘first-line’ investigations such as gastric aspirates and induced sputa, bronchoscopy may be worthwhile. Since TB may be rapidly progressive in HIV positive children, empirical therapy may be required more commonly than in immunocompetent children.

NON-TUBERCULOUS MYCOBACTERIAL RESPIRATORY INFECTIONS NTM may be identified in respiratory tract specimens by traditional microscopy, culture or ‘pan-mycobacterial’ NAD assays. These tests use primers complementary to gene sequences shared by most mycobacteria such as ribosomal

D. ANDRESEN RNA,77 and identify the species responsible for positive results by DNA sequencing of the amplification product and comparison to sequence databases. The utility of these assays in clinical practice has not been well defined. They may be help clarify the identity of the mycobacterial species being observed in patients with chronic lung disease, such as cystic fibrosis, who have positive acid-fast microscopy results. In immunosuppressed children with NTM lung infections, disseminated disease should be considered. Mycobacterial blood cultures, as well as biopsy of any potentially involved tissues for histology and culture, should be performed when clinically indicated in immunosuppressed children. The ATS has recently updated its position statement pertaining to NTM lung disease, which includes criteria which may be used to help ascertain the clinical significance of NTM in respiratory specimens. To be regarded as having NTM lung disease, patients should have pulmonary symptoms, nodules or cavities on CXR, or a high resolution CT scan showing multifocal bronchiectasis with multiple small nodules. They should also have laboratory evidence in the form of two positive sputa, one positive BAL specimen, or transbronchial or other lung biopsy showing granulomata or AFBs accompanied by a positive mycobacterial culture from any specimen.78 While the ATS criteria for NTM lung disease have not been specifically developed or validated for paediatric use, they have been widely adopted in a variety of clinical situations, including paediatric cystic fibrosis,1 and probably represent the best criteria presently available. It is worth noting that patients who have had NTM isolated from respiratory specimens but who do not meet these criteria should be observed for the development of additional features which might allow the diagnosis to be made, as the concept of NTM ‘colonization without disease’ is controversial.

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