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What steps do we need to take to improve diagnosis of tuberculosis in children? Expert Rev. Anti Infect. Ther. Early online, 1–16 (2015)
Elisabetta Venturini, Giulia Remaschi, Elettra Berti, Carlotta Montagnani, Luisa Galli, Maurizio de Martino* and Elena Chiappini Department of Health Sciences, Anna Meyer Children’s University Hospital, University of Florence, Florence, Italy *Author for correspondence: Tel.: +39 055 566 2494 Fax: +39 055 422 1012 [email protected]
Tuberculosis still represents a big global public health challenge. The diagnosis of tuberculosis and the differentiation between active and latent tuberculosis remain difficult, particularly in childhood, because of the lack of a gold standard test for diagnosis. In the last decade, novel diagnostic assays have been developed. Among immunologic tests, new assays based on the measurement of different cytokines released by specific T cells in response to Mycobacterium tuberculosis antigens, other than INF-g, have been investigated. Promising results rely on nucleic acid amplification techniques, also able to detect drugs resistance. Innovative research fields studied the modifications of CD27 expression in T cells as well as different host gene expression in response to M. tuberculosis. Further studies are needed to assess the diagnostic value and the accuracy of these new assays. KEYWORDS: children . diagnosis . IGRA . IP-10 . Mycobacterium tuberculosis . tuberculosis . Xpert MTB/RIF
The US WHO estimated that the tuberculosis (TB) burden in children was 550,000 cases in 2013, with up to 200 children dying of TB everyday [1]. Globally, about 6% of TB cases occur in children, whereas this proportion increases up to 10–20% in high-incidence settings [1]. In Europe, 2845 TB cases in children were reported the same year, accounting for 4.2% of all notified TB cases (range 0–7.8%) [2]. Moreover, no data are available on the burden of multi-drug-resistant TB in children. In 2013, WHO launched the Roadmap for childhood TB, with the goal of achieving zero TB deaths in children. One of the main areas of research and intervention addressed by the WHO is the development of new diagnostic tests, suitable for pediatric samples, for confirming TB diagnosis in children [1]. This systematic review provides an updated overview of the novel tests for TB diagnosis in children, their potential advantages with respect to the already available diagnostic tests, and their limitations. It especially focuses on future prospects in childhood TB diagnosis. Methods Search strategy
A literature search covering English language articles published between 1 January 2009 and
informahealthcare.com
10.1586/14787210.2015.1040764
31 December 2014 and concerning children only was conducted using multiple keywords and standardized terminology in PubMed database. Limits were field (Title/Abstract) and language (English). All relevant articles were then evaluated, and pertinent articles were included in this review. Study selection
The primary search was conducted by reviewing titles and abstracts of the studies, after that the full texts of eligible studies were screened for inclusion. An article was included in this review if it contained original data from children evaluating diagnostic tests for TB. Study eligibility, quality assessment and data extraction were checked for validity by a second author. All reference lists were also reviewed to identify pertinent publications and the websites of relevant government organizations and professional societies were consulted for documents of interest (WHO, Center for Disease Control and Prevention, European Center for Disease Control and Prevention, American Academy of Pediatrics, Health Protection Agency, UK Health Department). The search strategy has been included in Appendix 1.
2015 Informa UK Ltd
ISSN 1478-7210
1
Review
Venturini, Remaschi, Berti et al.
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Table 1. TST and IGRAs limitations
[3–14,17].
TST limitations
IGRAs limitations
Unable to distinguish between latent TB and TB disease
Unable to distinguish between latent TB and TB disease
Infiltrate measurement is operatordependent
More expensive
Not useful for monitoring TB treatment
Not useful for monitoring TB treatment
Double access to a healthcare facility
Blood samples must be processed within 8–30 h after collection
False positive results due to: . cross-reaction with BCG vaccinations . cross-reaction with non-tuberculous mycobacteria . booster effect
False positive results due to: . cross-reaction with non-tuberculous mycobacteria possible with few species
False negative results due to: . improper handling of the PPD solution or improper placement of the test . immunocompromised patients (HIV and other viral infections, severe malnutrition, disseminated TB disease, immunosuppressive drugs) . recent TB exposure (2–3 months delay in conversion)
False negative results due to: . immunocompromised patients (HIV and other viral infections, severe malnutrition, disseminated TB disease, immunosuppressive drugs) . children younger than 5 years
purified protein derivative (PPD). The reaction measurement may be operatordependent and the variability among experienced personnel is estimated to be roughly 15%, being much greater among inexperienced personnel and untrained people [6]. Additionally, the correct interpretation of the TST result requires an adequate evaluation of the patient’s risk factors: the cut-off for TST positivity is set at a higher level (10–15 mm induration) to optimize specificity for children lacking TB risk factors, whereas is set at a lower diameter (5 mm induration) to optimize sensitivity for high-risk children [3,4,6,7]. The TST presents several limitations to both the sensitivity and the specificity (TABLE 1). Unfortunately, children for whom the TST presents reduced sensitivity are those at increased risk of developing the severe form of TB disease. IFN-g release assays
IGRAs are diagnostic tests developed for the detection of TB infection measuring, IGRA: INF-g release assay; PPD: Purified protein derivative, TB: Tuberculosis, TST: Tuberculin skin test. ex vivo, the IFN-g released by CD4+ T cells in response to antigens found on Definitions the M. tuberculosis complex [3–6]. According to the most recent guideline recommendations, QuantiFERON-TB Gold-In tube (QFT-IT; Cellestis, Victoria, active TB is diagnosed in any child with Mycobacterium tuber- Australia) and T-SPOT.TB (T-SPOT; Immunotec, Oxford, culosis cultured or detected by microscopy or molecular meth- UK) are commercially available. The QFT is an enzyme-linked ods from sputum, gastric aspirate or other biologic samples [3]. immunosorbent assay whole blood test, and the result is Active TB diagnosis is also assigned to any child with clinical expressed as quantification of INF-g in IU per milliliter. The and radiological evidence of TB disease, and with either a his- test is positive when the INF-g response is over the cut-off of tory of exposure to an infectious case or a positive tuberculin 0.35 IU, after subtracting the negative control value from the skin test (TST)/IFN-g release assays (IGRA). In the absence of test antigen value. The T-SPOT is an enzyme-linked immunoa recognized gold standard, latent TB is diagnosed in a child sorbent spot assay performed on peripheral blood mononuclear with a positive TST and/or IGRA and no clinical, bacteriologi- cells, and the result is reported as the number of T cells procal or radiographic evidence of active TB [3]. ducing INF-g. When the number of spots in the test sample, after subtracting the number of spots in the negative control, The state of the art for the diagnosis of TB in exceeds a specific threshold, the result is considered positive. childhood There is no compelling evidence to support the use of one The diagnosis of latent and active TB in children is often diffi- IGRA over the other [6]. QTF-IT and T-SPOT assays use early cult and represents a challenge for pediatricians. Nowadays, it secreted antigenic target 6 (ESAT-6) and culture filter protein is mainly based on the combination of medical history investi- 10 (CFP-10) encoded by genes located within the region of gating TB contacts and symptoms consistent with TB (i.e., difference 1 locus of the M. tuberculosis genome [8]. The QFTchronic cough, fever, weight loss or failure to thrive), clinical IT uses a third antigen, TB7.7. The region of difference 1 antiexamination, radiological exams (i.e., chest x-ray), specific gens used in the IGRAs is not encoded in the genomes of immunological tests (TST/IGRAs) and bacteriological confir- Mycobacterium bovis-BCG strains and most species of nonmation, whenever is possible [3–7]. tuberculous mycobacteria, explaining why the IGRAs have an advantage over TST in identifying a natural M. tuberculosis Tuberculin skin test infection in settings with high non-tuberculous mycobacteria The TST method depends on the evidence that M. tuberculosis exposure and high BCG vaccination coverage [3,6,9–13]. Accordinfection causes a delayed-type hypersensitivity reaction to anti- ing to that, a general consensus exists concerning the greater genic components isolated from tubercle bacilli culture by specificity of the IGRAs than the TST in children [9–13]. doi: 10.1586/14787210.2015.1040764
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What steps do we need to take to improve diagnosis of tuberculosis in children?
Therefore, IGRA should be performed to reduce the number of false positive and unnecessary treatment in children who have received a BCG vaccine or have been exposed to nontuberculous mycobacteria. Otherwise, evidence from the available studies are contrasting and some reports suggest that IGRAs are not more sensitive than TST in identifying M. tuberculosis infection in children, particularly 95% in AFB microscopy positive respiratory specimens and approximately 50–80% in AFB-smear negative culture-positive specimens [30]. Currently, NAATs are still less sensitive than culture, considering also that the sensitivity of culture is relatively low. Moreover, the interpretation of data is complicated by the lack of test standardization and by the variable use of clinical diagnosis or culture as the reference standard. Therefore, there is still an ongoing debate on how to interpret positive NAATs in culturenegative specimens. Recently, the NAATs performance on non-pulmonary samples was evaluated for the diagnosis of pulmonary TB. A study on 456 Peruvian children compared PCR with culture in different specimens (nasopharyngeal aspirate, gastric aspirate and stool sample) for M. tuberculosis detection. The percentage of positive PCR was 62% in culture positive children; whereas it was 20% in culture negative ones. Overall, PCR sensitivity was 61.9%, specificity 79.7%, positive predictive value 33.3% and negative predictive value 92.7% when compared with cultures [21]. The authors conclude that PCR has an insufficient sensitivity and specificity for TB diagnosis in children, but in high-risk children, this test provides a rapid identification of around half culture positive cases [21]. Studies in adults showed wide variation in performance (7–100% sensitivity) of NAATs for detection of mycobacteria DNA fragments in urine samples. This process is based on the hypothesis that M. tuberculosis DNA could be released by lysed doi: 10.1586/14787210.2015.1040764
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Review
Venturini, Remaschi, Berti et al.
mycobacteria and filtered by the glomeruli, but no data are available in children. Few results are also reported concerning the performance of NAATs on blood specimens from children with TB, demonstrating a poor sensitivity (26.2%) of these assays [31]. Although several guidelines propose NAATs for M. tuberculosis detection in respiratory specimens [3,4,30], NAATs do not replace culture, which should always be performed. However, NAATs might be useful as a screening test for high-risk children because it rapidly identifies those who require treatment. The main limitations of NAATs are that these assays should not be used for treatment monitoring, because they are unable to distinguish live from dead bacilli, and their relative high costs [3,4,21,31]. Novel immunologic assays
Novel immunologic assays for the diagnosis of TB infection are under development, aiming to ameliorate the performance of currently available IGRAs. New immunologic tests may rely on novel mycobacterial antigens and/or evaluation of T-cell response measuring the release of other cytokines besides IFN-g. Assays based on novel M. tuberculosis T-cell antigens
In the literature, there is no consensus on the prognostic power of IGRAs in detecting progression to active TB [32,33]. Other antigens, other than ESAT-6, CFP-10 and TB7.7, have been studied in order to individuate a marker with higher predictive power [34]. In a study conducted in 846 household children, contacts of adults with active pulmonary TB, Dosanijh and colleagues investigated the T-cell response to Rv3873, Rv3878 and Rv3879c after M. tuberculosis exposure and established their prognostic value of progression to active TB. This study showed that T-cell responses to Rv3873 and Rv3878 predicted the progression to active TB with a similar prognostic power compared with CFP-10 and ESAT-6. Moreover, T-cell responses to Rv3873 and Rv3879c are early markers of TB exposure compared to ESAT-6/CFP-10 (p = 0.04) [34]. These findings suggest that 6.4% of children with a positive response to these antigens but a negative response to ESAT-6/CFP-10 might be infected with M. tuberculosis [34]. Novel host biomarkers in IGRAs supernatant IFN-g-inducible protein-10
A total of >40 biomarkers other than IFN-g are known to be expressed after antigens stimulation in IGRAs supernatant. The most promising one is the IFN-g-inducible protein (IP)-10. IP-10 is a small chemokine released by antigen-presenting cells involved in driving the pro-inflammatory immune response. Antigen-presenting cells express IP-10 in response to the interaction with T cells and other signals, mainly T cells derived [35]. In 2007, it was discovered that IP-10 is overexpressed in IGRA supernatants in patients with TB disease, but not in uninfected patients [36]. Nowadays, 10 studies have been done on this topic in both high and low endemic countries, including overall 2500 children (TABLE 2) [37–46]. IP-10 seems to have a similar diagnostic performance of IFN-g; however, its sensitivity ranges doi: 10.1586/14787210.2015.1040764
between 43 and 91% [35,37,42]. The use of IP-10 in distinguishing latent and active TB is controversial. Alsleben and colleagues showed no correlation between antigen-stimulated IP-10 median levels in active and latent TB (12.702 and 9.109 pg/ml, p = 0.24, respectively) [41]. Whittaker and colleagues found a higher level of IP-10 in children with latent TB compared with active TB [44]. In particular, the unstimulated IP-10 median level in patients with latent TB was 2.200 pg/ml, whereas it was 1.019 pg/ml in the active TB group (p = 0.018). The authors speculated that IP-10 levels could be lower in active TB patients due to an immunosuppressive effect of TB disease, where IP-10 levels might be higher in latent TB subjects because of a chronic inflammatory state [44]. However, this result was not statistically significant when comparing the stimulated IP-10 levels in the two groups (p = 0.85) [44]. Previous studies in adults confirmed non-univocal results [47–49]. Four studies enrolling >1000 children suggested that IP-10 concentration could not be influenced by age and immunodeficiency [38,41,43,45]. However, high IP-10 levels were increased also in other inflammatory diseases, such as lupus erythematous and thyroid disease, and infectious diseases, such as cerebral malaria, being not specific for TB detection [50]. Other biomarkers
Other markers, measured in IGRA supernatants, were investigated for the diagnosis of active TB or to discriminate between latent and active TB. The performance of the different markers in TB diagnosis has not been clearly demonstrated in the available studies [51–55]. Chegou and colleagues showed that IFN-a2, IL-1 receptor antagonist, soluble CD40 ligand and VEGF may be useful markers for TB disease diagnosis [51]. In particular, an increased level of CD40L was found in children with TB disease (p < 0.05), whereas a higher level of IL-1 receptor antagonist and IFN-a2 was detected in children without TB (p < 0.05). The authors also suggested that the most accurate markers in distinguishing active and latent TB are IL-1 receptor antagonist and IP-10 and VEGF (p = 0.03, p = 0.02 and p = 0.04, respectively) [51]. Kellar and colleagues observed that antigen-specific levels of IFN-g, IP-10, macrophage inflammatory protein-1b, monocyte chemoattractant protein (MCP)-1, TNF-a, IL-2, IL-6 and IL-8 in QFT-IT supernatants were significantly higher in patients with culture confirmed TB compared with controls. However, this study did not distinguish between biomarkers level in active and latent TB [52]. The combination of IL-5 and MCP-1 levels showed a good accuracy in discriminating between active and latent TB. In a study done on 70 subjects, IL-5 and MCP-1 levels accurately identified, respectively, 83% active and 88% latent TB cases [53]. However, the study has many limitations, including the small data set and the heterogeneity of test timing after treatment started [53]. Another study, by Lighter-Fisher and colleagues, evaluated cytokine levels after stimulation with QFT peptides, PPD peptides and recombinant ESAT-6 [54]. Median cytokine levels after stimulation with PPD were higher than those obtained with QFT peptides or Expert Rev. Anti Infect. Ther.
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Nigeria
Ruhwald et al. (2008)
Distinguish active and latent TB
Diagnosis of TB infection
Diagnosis of TB infection
Aim
49 children: 17 active TB . 16 latent TB . 16 controls .
120 children TB contact: . 59 at high risk of infection . 61 at low risk of infection
295 children TB contacts, after excluding indeterminate TST/ QFT-IT results: . 121 TST negative and QFT-IT negative . 98 TST positive and QFT-IT positive . 11 TST positive and QFT-IT negative . 17 TST negative and QFT-IT positive
Study subject
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA, X-Map)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
Release assay utilized (antigens; type of reader)
p = 0.01/ p > 0.3 IP-10 age-associated increase rate of positive response in low risk group, no correlation between IP-10 levels and age in high risk group
No significant difference of stimulated levels of IP-10 between active TB and latent TB
p = 0.85
p = 0.0085
p < 0.0001 Increasing test positivity rate by increasing smear grade among the TB contacts for all biomarkers
IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB and latent TB compared to controls
p < 0.0001
Significantly higher concentrations of the three biomarkers (IP-10, IL-2, IFN-g) were found in high risk children (smear positive contacts) than low risk ones (smear negative contacts and controls)
[44]
[40]
[39]
p < 0.001
IP-10 levels were higher in children TST positive/QFT-IT positive compared to children TST negative/QFT-IT negative Children with TST negative/QFTIT positive had higher IP-10 concentrations than children with TST positive/QFT-IT negative
Ref.
p-value
Results
[37–46].
CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
England
Brazil, Nepal
Petrucci et al. (2008)
Whittaker et al. (2008)
Country
Authors, (year)
Table 2. Studies assessing the performance of IP-10 dosage in QFT-IT supernatants in children
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What steps do we need to take to improve diagnosis of tuberculosis in children?
Review
doi: 10.1586/14787210.2015.1040764
Diagnosis of TB infection Distinguish active and latent TB
Diagnosis of latent TB infection
Ethiopia
India
Germany
Ethiopia
Yassin et al. (2011)
Syed Ahamed Kabeer et al. (2012)
Alsleben et al. (2012)
doi: 10.1586/14787210.2015.1040764
Yassin et al. (2013)
QFT-IT (ESAT-6, CFP10 and TB7.7; in-house ELISA)
48 children: 11 active TB . 14 latent TB . 8 non-tuberculous lymphadenopathy . 15 respiratory infection 486 children: 330 TB contacts . 156 controls .
.
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
135 children: 86 healthy controls . 59 healthy contacts .
p = 0.49
p < 0.001 Agreement of positive tests (IP-10, IFN-g and TST) was directly associated with a higher sputum bacilli grades
p = 0.24 IP-10 level showed no correlation with age
No differences in IP-10 levels in latent TB compared to active TB
IP-10 not correlated with age
p = 0.001/ p = 0.194
p > 0.1
No difference in IP10 levels were seen in HIV-infected and uninfected children Positivity of IP-10 was significantly higher than TST but not than QFT-IT in subjects £17 years of age
p = 0.8
p < 0.001
IP10 concentrations did not differ in children with confirmed TB and contacts
IP10 concentrations were higher in INF-g positive children independently of TST
[45]
[41]
[46]
[37]
p < 0.0001
The overall agreement between the QFT and IP-10 tests was high QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
p < 0.0001
IP-10 levels correlated with the risk of exposure to Mycobacterium tuberculosis in children
813 children: 322 with TB symptoms . 335 contacts . 156 controls .
[38]
p = 0.51
IP-10 expression is not agedependent
QFT -IT (not specified; ELISA, X-Map)
127 children: . 7 active TB . 12 high risk of TB . 87 low-moderate risk of TB . 21 no risk of TB
Ref.
(cont.). p-value
Results
Study subject
Release assay utilized (antigens; type of reader)
[37–46]
CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
Diagnosis of TB infection
Distinguish active and latent TB
Diagnosis of TB infection
USA
Lighter et al. (2009)
Aim
Country
Authors, (year)
Table 2. Studies assessing the performance of IP-10 dosage in QFT-IT supernatants in children
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Expert Rev. Anti Infect. Ther.
p < 0.005
p = 0.001
Combining QFT-IT and IP-10 assays the overall positive results improved significantly
IP-10 levels were significantly associated with age
QFT-IT, T.SPOT-TB (not specified; in-house ELISA, ELISPOT) 230 children: 12 active TB . 81 TB contacts . 137 controls Diagnosis of TB infection Distinguish active and latent TB Spain Latorre et al. (2014)
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CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
p = 0.023/ p = 0.012 The young age (
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What steps do we need to take to improve diagnosis of tuberculosis in children? Expert Rev. Anti Infect. Ther. Early online, 1–16 (2015)
Elisabetta Venturini, Giulia Remaschi, Elettra Berti, Carlotta Montagnani, Luisa Galli, Maurizio de Martino* and Elena Chiappini Department of Health Sciences, Anna Meyer Children’s University Hospital, University of Florence, Florence, Italy *Author for correspondence: Tel.: +39 055 566 2494 Fax: +39 055 422 1012 [email protected]
Tuberculosis still represents a big global public health challenge. The diagnosis of tuberculosis and the differentiation between active and latent tuberculosis remain difficult, particularly in childhood, because of the lack of a gold standard test for diagnosis. In the last decade, novel diagnostic assays have been developed. Among immunologic tests, new assays based on the measurement of different cytokines released by specific T cells in response to Mycobacterium tuberculosis antigens, other than INF-g, have been investigated. Promising results rely on nucleic acid amplification techniques, also able to detect drugs resistance. Innovative research fields studied the modifications of CD27 expression in T cells as well as different host gene expression in response to M. tuberculosis. Further studies are needed to assess the diagnostic value and the accuracy of these new assays. KEYWORDS: children . diagnosis . IGRA . IP-10 . Mycobacterium tuberculosis . tuberculosis . Xpert MTB/RIF
The US WHO estimated that the tuberculosis (TB) burden in children was 550,000 cases in 2013, with up to 200 children dying of TB everyday [1]. Globally, about 6% of TB cases occur in children, whereas this proportion increases up to 10–20% in high-incidence settings [1]. In Europe, 2845 TB cases in children were reported the same year, accounting for 4.2% of all notified TB cases (range 0–7.8%) [2]. Moreover, no data are available on the burden of multi-drug-resistant TB in children. In 2013, WHO launched the Roadmap for childhood TB, with the goal of achieving zero TB deaths in children. One of the main areas of research and intervention addressed by the WHO is the development of new diagnostic tests, suitable for pediatric samples, for confirming TB diagnosis in children [1]. This systematic review provides an updated overview of the novel tests for TB diagnosis in children, their potential advantages with respect to the already available diagnostic tests, and their limitations. It especially focuses on future prospects in childhood TB diagnosis. Methods Search strategy
A literature search covering English language articles published between 1 January 2009 and
informahealthcare.com
10.1586/14787210.2015.1040764
31 December 2014 and concerning children only was conducted using multiple keywords and standardized terminology in PubMed database. Limits were field (Title/Abstract) and language (English). All relevant articles were then evaluated, and pertinent articles were included in this review. Study selection
The primary search was conducted by reviewing titles and abstracts of the studies, after that the full texts of eligible studies were screened for inclusion. An article was included in this review if it contained original data from children evaluating diagnostic tests for TB. Study eligibility, quality assessment and data extraction were checked for validity by a second author. All reference lists were also reviewed to identify pertinent publications and the websites of relevant government organizations and professional societies were consulted for documents of interest (WHO, Center for Disease Control and Prevention, European Center for Disease Control and Prevention, American Academy of Pediatrics, Health Protection Agency, UK Health Department). The search strategy has been included in Appendix 1.
2015 Informa UK Ltd
ISSN 1478-7210
1
Review
Venturini, Remaschi, Berti et al.
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by CDL-UC San Diego on 06/01/15 For personal use only.
Table 1. TST and IGRAs limitations
[3–14,17].
TST limitations
IGRAs limitations
Unable to distinguish between latent TB and TB disease
Unable to distinguish between latent TB and TB disease
Infiltrate measurement is operatordependent
More expensive
Not useful for monitoring TB treatment
Not useful for monitoring TB treatment
Double access to a healthcare facility
Blood samples must be processed within 8–30 h after collection
False positive results due to: . cross-reaction with BCG vaccinations . cross-reaction with non-tuberculous mycobacteria . booster effect
False positive results due to: . cross-reaction with non-tuberculous mycobacteria possible with few species
False negative results due to: . improper handling of the PPD solution or improper placement of the test . immunocompromised patients (HIV and other viral infections, severe malnutrition, disseminated TB disease, immunosuppressive drugs) . recent TB exposure (2–3 months delay in conversion)
False negative results due to: . immunocompromised patients (HIV and other viral infections, severe malnutrition, disseminated TB disease, immunosuppressive drugs) . children younger than 5 years
purified protein derivative (PPD). The reaction measurement may be operatordependent and the variability among experienced personnel is estimated to be roughly 15%, being much greater among inexperienced personnel and untrained people [6]. Additionally, the correct interpretation of the TST result requires an adequate evaluation of the patient’s risk factors: the cut-off for TST positivity is set at a higher level (10–15 mm induration) to optimize specificity for children lacking TB risk factors, whereas is set at a lower diameter (5 mm induration) to optimize sensitivity for high-risk children [3,4,6,7]. The TST presents several limitations to both the sensitivity and the specificity (TABLE 1). Unfortunately, children for whom the TST presents reduced sensitivity are those at increased risk of developing the severe form of TB disease. IFN-g release assays
IGRAs are diagnostic tests developed for the detection of TB infection measuring, IGRA: INF-g release assay; PPD: Purified protein derivative, TB: Tuberculosis, TST: Tuberculin skin test. ex vivo, the IFN-g released by CD4+ T cells in response to antigens found on Definitions the M. tuberculosis complex [3–6]. According to the most recent guideline recommendations, QuantiFERON-TB Gold-In tube (QFT-IT; Cellestis, Victoria, active TB is diagnosed in any child with Mycobacterium tuber- Australia) and T-SPOT.TB (T-SPOT; Immunotec, Oxford, culosis cultured or detected by microscopy or molecular meth- UK) are commercially available. The QFT is an enzyme-linked ods from sputum, gastric aspirate or other biologic samples [3]. immunosorbent assay whole blood test, and the result is Active TB diagnosis is also assigned to any child with clinical expressed as quantification of INF-g in IU per milliliter. The and radiological evidence of TB disease, and with either a his- test is positive when the INF-g response is over the cut-off of tory of exposure to an infectious case or a positive tuberculin 0.35 IU, after subtracting the negative control value from the skin test (TST)/IFN-g release assays (IGRA). In the absence of test antigen value. The T-SPOT is an enzyme-linked immunoa recognized gold standard, latent TB is diagnosed in a child sorbent spot assay performed on peripheral blood mononuclear with a positive TST and/or IGRA and no clinical, bacteriologi- cells, and the result is reported as the number of T cells procal or radiographic evidence of active TB [3]. ducing INF-g. When the number of spots in the test sample, after subtracting the number of spots in the negative control, The state of the art for the diagnosis of TB in exceeds a specific threshold, the result is considered positive. childhood There is no compelling evidence to support the use of one The diagnosis of latent and active TB in children is often diffi- IGRA over the other [6]. QTF-IT and T-SPOT assays use early cult and represents a challenge for pediatricians. Nowadays, it secreted antigenic target 6 (ESAT-6) and culture filter protein is mainly based on the combination of medical history investi- 10 (CFP-10) encoded by genes located within the region of gating TB contacts and symptoms consistent with TB (i.e., difference 1 locus of the M. tuberculosis genome [8]. The QFTchronic cough, fever, weight loss or failure to thrive), clinical IT uses a third antigen, TB7.7. The region of difference 1 antiexamination, radiological exams (i.e., chest x-ray), specific gens used in the IGRAs is not encoded in the genomes of immunological tests (TST/IGRAs) and bacteriological confir- Mycobacterium bovis-BCG strains and most species of nonmation, whenever is possible [3–7]. tuberculous mycobacteria, explaining why the IGRAs have an advantage over TST in identifying a natural M. tuberculosis Tuberculin skin test infection in settings with high non-tuberculous mycobacteria The TST method depends on the evidence that M. tuberculosis exposure and high BCG vaccination coverage [3,6,9–13]. Accordinfection causes a delayed-type hypersensitivity reaction to anti- ing to that, a general consensus exists concerning the greater genic components isolated from tubercle bacilli culture by specificity of the IGRAs than the TST in children [9–13]. doi: 10.1586/14787210.2015.1040764
Expert Rev. Anti Infect. Ther.
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What steps do we need to take to improve diagnosis of tuberculosis in children?
Therefore, IGRA should be performed to reduce the number of false positive and unnecessary treatment in children who have received a BCG vaccine or have been exposed to nontuberculous mycobacteria. Otherwise, evidence from the available studies are contrasting and some reports suggest that IGRAs are not more sensitive than TST in identifying M. tuberculosis infection in children, particularly 95% in AFB microscopy positive respiratory specimens and approximately 50–80% in AFB-smear negative culture-positive specimens [30]. Currently, NAATs are still less sensitive than culture, considering also that the sensitivity of culture is relatively low. Moreover, the interpretation of data is complicated by the lack of test standardization and by the variable use of clinical diagnosis or culture as the reference standard. Therefore, there is still an ongoing debate on how to interpret positive NAATs in culturenegative specimens. Recently, the NAATs performance on non-pulmonary samples was evaluated for the diagnosis of pulmonary TB. A study on 456 Peruvian children compared PCR with culture in different specimens (nasopharyngeal aspirate, gastric aspirate and stool sample) for M. tuberculosis detection. The percentage of positive PCR was 62% in culture positive children; whereas it was 20% in culture negative ones. Overall, PCR sensitivity was 61.9%, specificity 79.7%, positive predictive value 33.3% and negative predictive value 92.7% when compared with cultures [21]. The authors conclude that PCR has an insufficient sensitivity and specificity for TB diagnosis in children, but in high-risk children, this test provides a rapid identification of around half culture positive cases [21]. Studies in adults showed wide variation in performance (7–100% sensitivity) of NAATs for detection of mycobacteria DNA fragments in urine samples. This process is based on the hypothesis that M. tuberculosis DNA could be released by lysed doi: 10.1586/14787210.2015.1040764
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Venturini, Remaschi, Berti et al.
mycobacteria and filtered by the glomeruli, but no data are available in children. Few results are also reported concerning the performance of NAATs on blood specimens from children with TB, demonstrating a poor sensitivity (26.2%) of these assays [31]. Although several guidelines propose NAATs for M. tuberculosis detection in respiratory specimens [3,4,30], NAATs do not replace culture, which should always be performed. However, NAATs might be useful as a screening test for high-risk children because it rapidly identifies those who require treatment. The main limitations of NAATs are that these assays should not be used for treatment monitoring, because they are unable to distinguish live from dead bacilli, and their relative high costs [3,4,21,31]. Novel immunologic assays
Novel immunologic assays for the diagnosis of TB infection are under development, aiming to ameliorate the performance of currently available IGRAs. New immunologic tests may rely on novel mycobacterial antigens and/or evaluation of T-cell response measuring the release of other cytokines besides IFN-g. Assays based on novel M. tuberculosis T-cell antigens
In the literature, there is no consensus on the prognostic power of IGRAs in detecting progression to active TB [32,33]. Other antigens, other than ESAT-6, CFP-10 and TB7.7, have been studied in order to individuate a marker with higher predictive power [34]. In a study conducted in 846 household children, contacts of adults with active pulmonary TB, Dosanijh and colleagues investigated the T-cell response to Rv3873, Rv3878 and Rv3879c after M. tuberculosis exposure and established their prognostic value of progression to active TB. This study showed that T-cell responses to Rv3873 and Rv3878 predicted the progression to active TB with a similar prognostic power compared with CFP-10 and ESAT-6. Moreover, T-cell responses to Rv3873 and Rv3879c are early markers of TB exposure compared to ESAT-6/CFP-10 (p = 0.04) [34]. These findings suggest that 6.4% of children with a positive response to these antigens but a negative response to ESAT-6/CFP-10 might be infected with M. tuberculosis [34]. Novel host biomarkers in IGRAs supernatant IFN-g-inducible protein-10
A total of >40 biomarkers other than IFN-g are known to be expressed after antigens stimulation in IGRAs supernatant. The most promising one is the IFN-g-inducible protein (IP)-10. IP-10 is a small chemokine released by antigen-presenting cells involved in driving the pro-inflammatory immune response. Antigen-presenting cells express IP-10 in response to the interaction with T cells and other signals, mainly T cells derived [35]. In 2007, it was discovered that IP-10 is overexpressed in IGRA supernatants in patients with TB disease, but not in uninfected patients [36]. Nowadays, 10 studies have been done on this topic in both high and low endemic countries, including overall 2500 children (TABLE 2) [37–46]. IP-10 seems to have a similar diagnostic performance of IFN-g; however, its sensitivity ranges doi: 10.1586/14787210.2015.1040764
between 43 and 91% [35,37,42]. The use of IP-10 in distinguishing latent and active TB is controversial. Alsleben and colleagues showed no correlation between antigen-stimulated IP-10 median levels in active and latent TB (12.702 and 9.109 pg/ml, p = 0.24, respectively) [41]. Whittaker and colleagues found a higher level of IP-10 in children with latent TB compared with active TB [44]. In particular, the unstimulated IP-10 median level in patients with latent TB was 2.200 pg/ml, whereas it was 1.019 pg/ml in the active TB group (p = 0.018). The authors speculated that IP-10 levels could be lower in active TB patients due to an immunosuppressive effect of TB disease, where IP-10 levels might be higher in latent TB subjects because of a chronic inflammatory state [44]. However, this result was not statistically significant when comparing the stimulated IP-10 levels in the two groups (p = 0.85) [44]. Previous studies in adults confirmed non-univocal results [47–49]. Four studies enrolling >1000 children suggested that IP-10 concentration could not be influenced by age and immunodeficiency [38,41,43,45]. However, high IP-10 levels were increased also in other inflammatory diseases, such as lupus erythematous and thyroid disease, and infectious diseases, such as cerebral malaria, being not specific for TB detection [50]. Other biomarkers
Other markers, measured in IGRA supernatants, were investigated for the diagnosis of active TB or to discriminate between latent and active TB. The performance of the different markers in TB diagnosis has not been clearly demonstrated in the available studies [51–55]. Chegou and colleagues showed that IFN-a2, IL-1 receptor antagonist, soluble CD40 ligand and VEGF may be useful markers for TB disease diagnosis [51]. In particular, an increased level of CD40L was found in children with TB disease (p < 0.05), whereas a higher level of IL-1 receptor antagonist and IFN-a2 was detected in children without TB (p < 0.05). The authors also suggested that the most accurate markers in distinguishing active and latent TB are IL-1 receptor antagonist and IP-10 and VEGF (p = 0.03, p = 0.02 and p = 0.04, respectively) [51]. Kellar and colleagues observed that antigen-specific levels of IFN-g, IP-10, macrophage inflammatory protein-1b, monocyte chemoattractant protein (MCP)-1, TNF-a, IL-2, IL-6 and IL-8 in QFT-IT supernatants were significantly higher in patients with culture confirmed TB compared with controls. However, this study did not distinguish between biomarkers level in active and latent TB [52]. The combination of IL-5 and MCP-1 levels showed a good accuracy in discriminating between active and latent TB. In a study done on 70 subjects, IL-5 and MCP-1 levels accurately identified, respectively, 83% active and 88% latent TB cases [53]. However, the study has many limitations, including the small data set and the heterogeneity of test timing after treatment started [53]. Another study, by Lighter-Fisher and colleagues, evaluated cytokine levels after stimulation with QFT peptides, PPD peptides and recombinant ESAT-6 [54]. Median cytokine levels after stimulation with PPD were higher than those obtained with QFT peptides or Expert Rev. Anti Infect. Ther.
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Nigeria
Ruhwald et al. (2008)
Distinguish active and latent TB
Diagnosis of TB infection
Diagnosis of TB infection
Aim
49 children: 17 active TB . 16 latent TB . 16 controls .
120 children TB contact: . 59 at high risk of infection . 61 at low risk of infection
295 children TB contacts, after excluding indeterminate TST/ QFT-IT results: . 121 TST negative and QFT-IT negative . 98 TST positive and QFT-IT positive . 11 TST positive and QFT-IT negative . 17 TST negative and QFT-IT positive
Study subject
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA, X-Map)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
Release assay utilized (antigens; type of reader)
p = 0.01/ p > 0.3 IP-10 age-associated increase rate of positive response in low risk group, no correlation between IP-10 levels and age in high risk group
No significant difference of stimulated levels of IP-10 between active TB and latent TB
p = 0.85
p = 0.0085
p < 0.0001 Increasing test positivity rate by increasing smear grade among the TB contacts for all biomarkers
IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB and latent TB compared to controls
p < 0.0001
Significantly higher concentrations of the three biomarkers (IP-10, IL-2, IFN-g) were found in high risk children (smear positive contacts) than low risk ones (smear negative contacts and controls)
[44]
[40]
[39]
p < 0.001
IP-10 levels were higher in children TST positive/QFT-IT positive compared to children TST negative/QFT-IT negative Children with TST negative/QFTIT positive had higher IP-10 concentrations than children with TST positive/QFT-IT negative
Ref.
p-value
Results
[37–46].
CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
England
Brazil, Nepal
Petrucci et al. (2008)
Whittaker et al. (2008)
Country
Authors, (year)
Table 2. Studies assessing the performance of IP-10 dosage in QFT-IT supernatants in children
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What steps do we need to take to improve diagnosis of tuberculosis in children?
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doi: 10.1586/14787210.2015.1040764
Diagnosis of TB infection Distinguish active and latent TB
Diagnosis of latent TB infection
Ethiopia
India
Germany
Ethiopia
Yassin et al. (2011)
Syed Ahamed Kabeer et al. (2012)
Alsleben et al. (2012)
doi: 10.1586/14787210.2015.1040764
Yassin et al. (2013)
QFT-IT (ESAT-6, CFP10 and TB7.7; in-house ELISA)
48 children: 11 active TB . 14 latent TB . 8 non-tuberculous lymphadenopathy . 15 respiratory infection 486 children: 330 TB contacts . 156 controls .
.
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
135 children: 86 healthy controls . 59 healthy contacts .
p = 0.49
p < 0.001 Agreement of positive tests (IP-10, IFN-g and TST) was directly associated with a higher sputum bacilli grades
p = 0.24 IP-10 level showed no correlation with age
No differences in IP-10 levels in latent TB compared to active TB
IP-10 not correlated with age
p = 0.001/ p = 0.194
p > 0.1
No difference in IP10 levels were seen in HIV-infected and uninfected children Positivity of IP-10 was significantly higher than TST but not than QFT-IT in subjects £17 years of age
p = 0.8
p < 0.001
IP10 concentrations did not differ in children with confirmed TB and contacts
IP10 concentrations were higher in INF-g positive children independently of TST
[45]
[41]
[46]
[37]
p < 0.0001
The overall agreement between the QFT and IP-10 tests was high QFT-IT (ESAT-6, CFP10 and TB7.7; ELISA)
p < 0.0001
IP-10 levels correlated with the risk of exposure to Mycobacterium tuberculosis in children
813 children: 322 with TB symptoms . 335 contacts . 156 controls .
[38]
p = 0.51
IP-10 expression is not agedependent
QFT -IT (not specified; ELISA, X-Map)
127 children: . 7 active TB . 12 high risk of TB . 87 low-moderate risk of TB . 21 no risk of TB
Ref.
(cont.). p-value
Results
Study subject
Release assay utilized (antigens; type of reader)
[37–46]
CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
Diagnosis of TB infection
Distinguish active and latent TB
Diagnosis of TB infection
USA
Lighter et al. (2009)
Aim
Country
Authors, (year)
Table 2. Studies assessing the performance of IP-10 dosage in QFT-IT supernatants in children
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Expert Rev. Anti Infect. Ther.
p < 0.005
p = 0.001
Combining QFT-IT and IP-10 assays the overall positive results improved significantly
IP-10 levels were significantly associated with age
QFT-IT, T.SPOT-TB (not specified; in-house ELISA, ELISPOT) 230 children: 12 active TB . 81 TB contacts . 137 controls Diagnosis of TB infection Distinguish active and latent TB Spain Latorre et al. (2014)
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CFP-10: Culture filter protein 10; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunosorbent spot; ESAT-6: Early secreted antigenic target 6; IP-10: IFN-g-inducible protein-10; QFT-IT: QuantiFERON-TB Gold In-Tube; TB: Tuberculosis; TST: Tuberculosis skin test.
p = 0.023/ p = 0.012 The young age (
