Published online January 2, 2007
PEDIATRICS Vol. 119 No. 1 January 2007, pp. e1-e5 (doi:10.1542/10.1542/peds.2006-1057)

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EXPERIENCE & REASON

T-Cell–Based Diagnosis of Neonatal Multidrug-Resistant Latent Tuberculosis Infection

Luca Richeldi, MD, PhD^a, Katie Ewer, PhD^b, Monica Losi, PhD^a, Barbara M. Bergamini, MD^a, Kerry Millington, BSc^b,c, Leonardo M. Fabbri, MD^a and Ajit Lalvani, FRCP, DM^b,c

^a Departments of Respiratory Disease and Pediatrics, University of Modena and Reggio Emilia, Modena, Italy
^b Tuberculosis Immunology Group, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
^c Tuberculosis Immunology Group, Department of Respiratory Medicine, National Heart and Lung Institute, Wright Fleming Institute of Infection and Immunity, Imperial College London, London, United Kingdom

ABSTRACT

Young children exposed to tuberculosis have a high risk of progression to severe tuberculosis disease, but diagnosis of recent infection is hindered by the poor sensitivity of the tuberculin skin test. Whether new blood tests can detect latent infection in this vulnerable group is unknown because there is no gold standard. We monitored a tuberculin skin test–negative infant whose mother had infectious multidrug-resistant tuberculosis with enzyme-linked immunospot, a blood test that enumerates Mycobacterium tuberculosis–specific T cells. The enzyme-linked immunospot test became persistently positive by 6 months, and 18 months later the child developed active tuberculosis despite appropriate chemoprophylaxis. At this point, the magnitude of the enzyme-linked immunospot response increased >10-fold. Our findings demonstrate that this blood test detected latent infection with dormant, yet viable, bacilli and illustrate how enzyme-linked immunospot could improve diagnosis of childhood tuberculosis infection.

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Key Words: tuberculosis • neonate • diagnosis • ELISpot

Abbreviations: LTBI, latent tuberculosis infection • TST, tuberculin skin test • ELISpot, enzyme-linked immunospot • MDR, multidrug-resistant • PPD, purified protein derivative • ESAT-6, early secretory antigenic target 6 • CFP10, culture filtrate protein 10 • SKSD, streptokinase-streptodornase • SFC, spot-forming cell

The current strategy of targeted tuberculin testing and treatment of latent tuberculosis infection (LTBI) is aimed at high-risk groups, including HIV-infected people, people on immunosuppressive medication, and young children.¹ Unfortunately, it is precisely these groups in whom the tuberculin skin test (TST) is least reliable, because the results are frequently false-negative.² The most vulnerable group comprises infants aged under 1 year, whose risk of progression after Mycobacterium tuberculosis infection is >50%.^3,4 The TST can be falsely negative in active tuberculosis and LTBI, but the false-negative rate in LTBI cannot be calculated because of the absence of a gold-standard test.⁵ A recent review of neonatal exposure to tuberculosis in North American neonatal units found that none of the 2635 newborns developed a positive skin test.⁶ We believe that it is unlikely that none of the newborns would have acquired infection and assume that the lack of any positive skin-test results suggests that the TST has low sensitivity in this age group.

New rapid blood tests, based on detection of interferon- released by T cells in response to M tuberculosis antigens, offer a novel approach to diagnosing LTBI.⁷ An enzyme-linked immunospot (ELISpot) assay that detects individual interferon-–producing T cells (T-SPOT.TB, Oxford Immunotec Ltd, Abingdon, United Kingdom) is approved for use in Europe and Canada and is being evaluated by the US Food and Drug Administration; this assay has higher diagnostic sensitivity than the TST in children with active tuberculosis, particularly children younger than 3 years.⁸ Moreover, in a large school tuberculosis outbreak, ELISpot results correlated more closely with M tuberculosis exposure than the Heaf intradermal test, suggesting that it is also more sensitive for detecting LTBI.⁹ However, in the absence of a gold-standard test for latent infection, it is difficult to confirm whether infants, and other high-risk groups, with a positive ELISpot results and a negative TST result truly have LTBI. Only progression to active tuberculosis can provide definitive evidence that ELISpot detects LTBI when the TST fails to do so, but there are no reported examples of this. Here we report the application of ELISpot for diagnosis of LTBI in a neonate born to a mother with untreated multidrug-resistant (MDR) tuberculosis.

CASE REPORT

A 24-year-old woman from Moldova delivered a healthy infant at the University Hospital of Modena, Italy. One week after delivery, chest radiography strongly suggested active tuberculosis (Fig 1A). Three sputum samples were strongly positive for acid-fast bacilli. Standard 4-drug antituberculous therapy was started, and she was separated from her infant. Three weeks later, her sputum specimens grew M tuberculosis that was resistant to isoniazid and rifampin but sensitive to pyrazinamide and ethambutol. After switching to a 5-drug regimen (pyrazinamide, moxifloxacin, ethambutol, streptomycin, and clofazimine), the woman progressively improved.

View larger version (112K): [in this window] [in a new window]   FIGURE 1 A, Chest radiograph of the index case. Extensive patchy consolidation with areas of cavitation is seen in the middle and upper zones, consistent with active tuberculosis. B, Chest radiograph of the child at 24 months of age, the time at which he presented with fever and respiratory symptoms, showing right-sided hilar lymphadenopathy and ipsilateral parenchymal compression. C, Molecular strain typing (DNA fingerprinting) using insertion sequence 6110 restriction fragment length polymorphism (RFLP) analysis (IS 6110-based RFLP) of the MDR M tuberculosis isolates obtained from the mother's sputum in November 2001 (upper) and from her child's gastric aspirate in December 2003 (lower lane). The IS6110 RFLP patterns are identical, indicating that the mother and child were infected with the same strain.

 
Her asymptomatic 4-week-old infant had been intensively exposed during the 11 days from birth until the mother's hospital admission. Chest radiography and mediastinal ultrasound were normal, and the TST was negative. The infant did not receive BCG vaccination and was given 9 months of chemoprophylaxis with ethambutol (15 mg/kg daily) and pyrazinamide (20 mg/kg daily) with directly observed therapy, consistent with Centers for Disease Control and Prevention guidelines.¹⁰ Recognizing that the TST result might be false-negative, we offered supplementary ELISpot testing within a protocol approved by the Modena University Hospital Institutional Review Board, and the parents provided informed consent. Accordingly, the child had an ELISpot test, TST, and clinical review at the age of 11 weeks and again at 6, 12, 18, and 24 months of age.

METHODS

The TST was administered by the Mantoux method using 5 IU of purified protein derivative-Siebert (Biocine Test PPD, Chiron Corporation/Sclavo, Siena, Italy). PPD-Siebert (or tuberculin PPD, lot number 49608) is the US standard for all PPD preparations, which are standardized against this product. The transverse diameter of cutaneous induration was measured with a ruler and recorded 72 hours after inoculation, using 5 mm as the cutoff for a positive test.² TSTs were placed and read by an experienced pulmonologist who routinely looks after patients with tuberculosis and who was blinded to the ELISpot results. The cutaneous appearance of peau d'orange was noted, confirming intradermal inoculation of PPD. No specific measures were used to prevent terminal rounding of the induration measurements. The ELISpots were performed as described previously by using recombinant early secretory antigenic target 6 (ESAT-6), recombinant culture filtrate protein 10 (CFP10), and 6 pools of overlapping peptides derived from these antigens (3 pools of peptides for each antigen).¹¹ Mitogen-stimulated positive control wells contained phytohemagglutinin (ICN Biomedicals, Solon, OH), and antigen-stimulated positive control wells contained streptokinase-streptodornase (SKSD; Wyeth Farma SA, Madrid, Spain) as an index of M tuberculosis–unrelated antigen-specific T-cell responsiveness. ELISpot plates were scored by an automated counter (AID-GmbH, Strasburg, Germany).¹¹ All test conditions were in duplicate wells, each of which contained 250000 peripheral blood mononuclear cells. Pairs of wells containing ESAT-6 or CFP10 antigen were scored positive if they contained a mean of at least 10 spot-forming cells (SFCs) more than the mean of the negative control wells, as previously described^8,9,12 (this threshold is shown as a dashed line in Fig 2). The cutoff for a positive response to ESAT-6- and CFP10-derived peptide pools was 5 SFCs above background, as previously described.^8,9,12 The commercially available diagnostic test T-SPOT.TB is based on the ELISpot assay used here and in our earlier studies,^{8,9,11,13–15} but it provides the 3 peptide pools for ESAT-6 and the 3 peptide pools for CFP10 in 1 test well each; the test does not use recombinant antigens.

View larger version (12K): [in this window] [in a new window]   FIGURE 2 Time course of ELISpot and TST results during the first 3 years of life. Shown are ELISpot assay results indicating responses to ESAT-6 (white bars) and CFP10 (black bars) antigens for the child at all time points. The ELISpot result first became positive at 6 months of age and remained positive thereafter. Responses in negative control wells were <3 SFCs at all time points, and all ESAT-6 and CFP10 responses shown are after subtraction of corresponding negative control values. Mitogen-stimulated positive control wells were positive at a level of >100 SFCs at all time points and were not significantly higher at 24 months than at earlier time points. SKSD was used as an index of M tuberculosis–unrelated antigen-specific T-cell responsiveness. The response to SKSD first became positive at 6 months (at a level of 25 SFCs) and remained positive thereafter, and the level of response was not significantly higher at 24 months (26 SFCs) than at earlier time points. Responses to peptide pools were positive for ESAT-6–derived peptides at 12 months of age (the summated response to each of the 3 ESAT-6 peptide pools was 16 SFCs, and the summated response to each of the 3 CFP10 peptide pools was 16 SFCs) and again at 24 months (the summated ESAT-6 peptide response was 122 SFCs, and the summated CFP10 peptide response was 136 SFCs). The reason for a lack of response to CFP10 antigen at 18 months is unclear at this time. T-cell responses to ESAT-6 and CFP10 are often discordant, which is why both antigens, or peptides from both antigens, need to be included in diagnostic immunoassays.

 

RESULTS

Results of the first ELISpot assay and a repeat TST, performed at 11 weeks of age after 7 weeks of chemoprophylaxis, were negative (Fig 2). At 6 months of age, the ELISpot result was positive and remained positive at a similar level at 12 and 18 months of age, although the TST results remained negative (Fig 2). At 24 months of age, the child presented with a 2-week history of fever, night sweats, and cough. ELISpot results again were positive but with a 10-fold higher response than previously (Fig 2). Responses to phytohemagglutinin and SKSD were at a similar level to earlier time points (data not shown), indicating that the increased response to M tuberculosis was not attributable to a nonspecific increase in immune responsiveness. The TST result was then positive at 10 mm induration, and chest radiography was consistent with active pulmonary tuberculosis (Fig 1B). The next day, the child underwent bronchoalveolar lavage, which revealed acid-fast bacilli. The child commenced on the same 5 antituberculous drugs as the mother. Progressive clinical and radiologic improvement ensued, and 8 weeks later, M tuberculosis was cultured from lavage fluid. The drug-resistance pattern was the same as the mother's isolate, and DNA fingerprinting indicated that his isolate was identical to that of his mother (Fig 1C). In parallel with the child's clinical improvement during treatment, the magnitude of the ELISpot response gradually declined (Fig 2).

DISCUSSION

The child was intensely exposed to MDR M tuberculosis for the first 11 days after birth and was started on a 9-month course of prophylactic ethambutol and pyrazinamide at 4 weeks under a regimen of directly observed therapy. Results of all 4 TSTs during the first 18 months of life were negative, but ELISpot results were positive by 6 months of age and remained persistently positive thereafter. The subsequent development of active tuberculosis confirmed that the positive ELISpot results during the preceding 18 months reflected true LTBI in the context of repeatedly negative skin test results. This is the first reported diagnosis of LTBI in an infant by means other than the TST. The reason for the initial negative ELISpot assay result at 11 weeks is uncertain but may be because the child had already received 7 weeks of treatment with pyrazinamide and ethambutol, which might have suppressed mycobacterial replication. It is unlikely that the positive ELISpot result was induced by inoculation of PPD during the initial TST, because repeated testing with the TST does not induce false-positive ELISpot results.¹⁶ Although there is, in general, an excellent correlation between ELISpot responses to recombinant ESAT-6 and CFP10 antigens and their respective peptide pools,^13,17 a response to peptide pools was first detected in our patient 12 months after birth. Because T-SPOT.TB (which was not commercially available at the time of this study) contains peptide pools but not recombinant antigens, results would presumably have become positive 12 months after birth.

The ELISpot assay read-out is quantitative and dynamic because it enumerates effector-memory T cells that recently encountered M tuberculosis antigens in vivo.¹⁸ Interpretation of ELISpot results as a continuous, quantitative measure of the host immune response might, therefore, provide clinically useful information about the dynamic host-pathogen equilibrium in vivo. It is interesting that the number of these cells increased considerably when the child developed active tuberculosis. A major determinant of the frequency of antigen-specific T cells in vivo is antigen load, which is closely related to pathogen burden.¹⁸ Thus, the increased ELISpot response at the time of symptom onset probably reflects increasing bacterial burden in vivo as the latent infection reactivated. Only at this point was the cellular immune response to M tuberculosis large enough to provide a positive TST result. The subsequent progressive decline in the ELISpot response with antituberculosis treatment is consistent with the response to treatment previously noted in adults^14,18 and raises the possibility that ELISpot could be used to monitor an individual's response to treatment in some cases.¹⁹

Could active tuberculosis at 24 months have resulted from recent reinfection rather than reactivation of perinatal LTBI? This is unlikely for the following reasons. First, the DNA fingerprint of the child's M tuberculosis strain was identical to that of his mother. Second, given the infant's limited social interaction, infection after the immediate perinatal period could only have been acquired from household members comprising the mother, father, and paternal grandparents. The mother was separated from her child 11 days after birth, and contact resumed only after 3 months of treatment when her sputum cultures were negative. The child's father was diagnosed with subclinical active MDR tuberculosis at screening when the infant was aged 11 weeks; he was treated before development of cough and was not infectious at any point.²⁰ His isolate had the same DNA fingerprint as the mother's strain. Active tuberculosis was clinically and radiographically excluded in both grandparents.

MDR tuberculosis is a growing problem globally, and the management of contacts at risk of exposure to MDR M tuberculosis is a major clinical challenge.²¹ One nonrandomized, uncontrolled study²² suggested significant benefit from chemoprophylaxis in child contacts of adult MDR tuberculosis using drugs to which the index case's strains were susceptible. Current Centers for Disease Control and Prevention guidelines recommend 9 to 12 months of treatment with pyrazinamide and ethambutol for children and infants who are exposed to rifampicin- and isoniazid-resistant M tuberculosis¹⁰; it is noteworthy that 9 months of treatment did not prevent active tuberculosis in our patient. Because our results pertain to a child who was exposed to MDR tuberculosis, they might not necessarily be generalizable to drug-sensitive tuberculosis. However, to date, the performance of ELISpot in contacts of drug-sensitive tuberculosis has been very similar to its performance in contacts of MDR tuberculosis.^11,20,23

This report suggests that ELISpot may be a useful tool for diagnosis of LTBI in young children with recent tuberculosis exposure. Future studies should aim to quantify the predictive value of a positive ELISpot result for subsequent development of active tuberculosis in young children and other high-risk groups. Such studies could also assess whether dynamic changes in the magnitude of the ELISpot response can serve as an early marker of progression to incipient active disease before the onset of symptoms.

ACKNOWLEDGMENTS

This work was supported by the Wellcome Trust. Dr Lalvani is a Wellcome Senior Research Fellow in Clinical Science.

We thank Andrea Gori (University of Milano, Milano, Italy) for restriction fragment length polymorphism typing; Patrizia Marchegiano (Policlinico Hospital, Modena, Italy) for help with project management; Adam Whalen (VLA, Weybridge, United Kingdom) and Mahavir Singh (Braunschweig, Germany) for the kind gift of recombinant antigens; and Ben Marais (University of Stellenbosch, Cape Town, South Africa) and Paul Heath (St George's Hospital Medical School, London, United Kingdom) for critical evaluation of the manuscript.

FOOTNOTES

Accepted Aug 2, 2006.

Address correspondence to Ajit Lalvani, FRCP, DM, Tuberculosis Immunology Group, Wright Fleming Institute of Infection and Immunity, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1NY, United Kingdom. E-mail: ajit.lalvani{at}ndm.ox.ac.uk

Financial Disclosure: Dr Lalvani is a named inventor on patents relating to T-cell–based diagnosis filed by the University of Oxford. Regulatory approval and commercialization of ELISpot (T-SPOT.TB) has been undertaken by a spin-off company of the University of Oxford (Oxford Immunotec Ltd, Oxford, England), in which Dr Lalvani has a share of equity and to which he acts as scientific advisor in a nonexecutive capacity. Dr Ewer is a named inventor on a patent application relating to the application of ELISpot filed by the University of Oxford. The University of Oxford has a share of equity in Oxford Immunotec Ltd.

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