Latent tuberculosis infection and interferon-gamma release assays: what new knowledge did we gain through the Journal in 2009?

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1 INT J TUBERC LUNG DIS 14(12): The Union 2009 YEAR IN REVIEW Latent tuberculosis infection and interferon-gamma release assays: what new knowledge did we gain through the Journal in 2009? M. J. van der Werf, F. van Leth Research Unit, KNCV Tuberculosis Foundation, The Hague, and Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands BOTH in vivo and in vitro tests are applied for the diagnosis of latent tuberculosis (TB) infection and for assessing the annual risk of infection in epidemiological studies. Both types of tests have their advantages and disadvantages. The 2009 issues of the International Journal of Tuberculosis and Lung Disease published studies that discuss tests for TB infection in different populations and for different purposes. Below we discuss these studies and relate them to other recent publications on the same topic. TUBERCULIN SKIN TEST SURVEYS Tuberculin skin test (TST) surveys are used for estimating the prevalence of infection and annual risk of tuberculosis infection (ARTI) in populations. The ARTI was used to estimate the incidence of sputum smear-positive TB applying the Styblo rule, 1 which assumed a fixed mathematical relationship between the incidence of smear-positive TB, the prevalence of smear-positive TB and the ARTI. This mathematical relationship is no longer considered to be valid. 2 Today, TST surveys are mainly considered useful for assessing time trends and geographical variations in risk of infection. 3 Three studies reported the results of TST surveys in schoolchildren. 4 6 Bachtiar et al. reported on single TST surveys in three provinces in Indonesia. The same authors reported on a similar study in another province in Indonesia in The absolute level of the ARTI reported in these studies is not very informative, as it highly depends on the analysis method chosen to estimate the prevalence of infection. 8,9 The studies did allow for a geographical comparison, with North Sulawesi having a higher ARTI (using mirror method with mode at 15 mm) than Central Java, East Nusa Tenggara or West Sumatra. Both publications from 2009 concluded that intensification of TB control efforts is needed in Indonesia to reduce the transmission of TB. 4,5 Al-Asbi et al. compared the results of a TST survey conducted in Yemen in 1991 with a tuberculin survey conducted in Their conclusion is that TB infection seems to be declining considerably. The 2007 survey results proved difficult to analyse and to interpret due to TST distributions without a clear mode or antimode. It is not possible to predict beforehand in which situation tuberculin surveys will give data that can be analysed and interpreted and in which situations this will prove to be difficult. A study in South Africa among gold miners determined the prevalence of latent TB infection (LTBI) using the TST, and assessed risk factors for a positive TST. 10 The prevalence of LTBI was very high in this population (89%). Factors independently associated with a TST < 10 mm were positive human immunodeficiency virus (HIV) status and not working underground. This study showed a clear frequency distribution of the indurations and a mode at 12 mm. In the analysis of the TST survey in Yemen, only non bacille Calmette-Guérin (BCG) vaccinated children were included. For the studies in Indonesia, the results of BCG-vaccinated and non-bcg-vaccinated children were reported separately and combined. In the Yemen study, 41% of the children had a BCG scar; in the three provinces in Indonesia, Nusa Tenggara, Central Java and North Sulawesi, respectively 52%, 87% and 79% had a BCG scar. The population-based TST survey among gold miners in South Africa reported that the distribution of TST responses was similar in BCG-vaccinated and non-vaccinated individuals, with 266 of 429 (62%) having a BCG scar. 10 In 2006, the results of an extensive literature review and meta-analysis assessing the effect of BCG vaccination on TST were reported in the Journal. 11 The authors concluded that the effect on TST of BCG received in infancy is minimal, especially 10 years or more after vaccination. However, BCG received after infancy produces more frequent, more persistent and larger TST reactions. As many countries will now have Correspondence to: Marieke J van der Werf, Unit Research, KNCV Tuberculosis Foundation, P O Box 146, The Hague 2501 CC, The Netherlands. Tel: (+31) Fax: (+31) vanderwerfm@kncvtbc.nl

2 1526 The International Journal of Tuberculosis and Lung Disease a BCG coverage of >80%, 12 the non-bcg-v accinated population will likely not be representative of the total study population. It seems prudent, therefore, to include BCG-vaccinated and non-bcg-vaccinated individuals in TST surveys and to assess whether there is a difference in the TST response, or to report TST positivity separately for BCG-vaccinated and non-vaccinated individuals, and to combine the two groups where possible. This is indeed what is done in the most recent surveys. 8,13 14 Another variable present in TST surveys is the type of tuberculin used. Sgountzos et al. showed that there was no difference between RT23 (Staten Serum Institute, Copenhagen, Denmark) and Merieux tuberculin (Mérieux Biological Institute, Lyons, France) when testing healthy police academy students in Athens. 15 Data comparing different tuberculin reagents are available from older studies These studies show varying results. The different tuberculin reagents may give comparable results or many discrepant results. The recommendation to use the same tuberculin in successive TST surveys thus still holds. 19 INDIRECT AND DIRECT MEASUREMENT OF ARTI TST surveys provide an indirect measurement of the ARTI. Del Rio Camacho et al. compared the indirect method used in TST surveys with a direct method of measuring ARTI, which is a formal incidence study. 20 For the direct method, schoolchildren with a negative TST result were retested 1 year after the initial test. The authors discuss the limited possibility of boosting, although they did not include BCG-vaccinated children. Of the following criteria that are used for considering a second TST to be positive 5 mm, >10 mm, 10 mm, or a difference of at least 6 mm or 12 mm with respect to the diameter of the preceding induration an increase of >10 mm between the first and second TST provided an ARTI that corresponded best to the ARTI obtained using the indirect method. IMMUNOSUPPRESSION AND TST Two studies used the TST for testing individuals with a risk factor for TB, i.e., diabetes and persons living with HIV/acquired immune-deficiency syndrome (PLWHAs). 21,22 Vitek et al. reported that 26% of PLWHAs tested in Orel Oblast in Russia had a positive TST result. This is a minimum prevalence, as HIV-infected individuals often lose TST sensitivity, which is predominantly attributable to anergy. 23 The authors recommend that the TB control programme in Russia implement TST screening among PLWHAs. Webb et al. tested children and adolescents with type 1 diabetes in the Western Cape Province in South Africa. 22 They report a high prevalence of TB disease (3488/ ), and recommend routine TB screening of children and adolescents in the Cape Province. INTERFERON-GAMMA RELEASE ASSAYS As there is no gold standard for TB infection, the evaluation of (new) diagnostic tests for TB infection remains a matter of controversy, although these tests are used in a wide variety of situations, including individual patient management, contact investigations and serial testing for individuals in high-risk settings for infection. Different settings will have different effects on the interpretation of the test results. This was shown in a study by O Neal et al., where a second generation interferon-gamma release assay (IGRA) was used in a contact investigation with proven TB transmission. 24 The authors rightly point out the negative predictive value of the test and its relationship with the assumed prevalence of TB infection in the study population, and conclude that the use of a QuantiFERON -TB (QFT) test in settings with a high pre-test risk of infection maybe suboptimal. Taking the assumed background prevalence of infection into account when interpreting test results is therefore recommended. A much debated issue around the use of IGRAs is the occurrence of conversions and reversions in the setting of serial testing, as was also included in a research agenda for T-cell-based assays published in 2007 by Pai et al. 25 Some of the questions were addressed in a study by Pai et al. concerning serial assessment of infection with the QuantiFERON -TB Gold (QFT-G) test in a household contact study. 26 It showed, not surprisingly, that the rate of conversion and reversion differed for different definitions of conversion and reversion. This needs to be taken into account when the tests are being used in community surveys where sample sizes are large and small differences between definitions are being amplified. QFT-G reversions were more likely when the QFT-G test at baseline was only weakly positive, giving rise to a change in test result when only minor changes around the QFT-G cut-off point take place. Such minor changes are quite possible given the substantial within-subject variation of the QFT-G test. In a systematic review, van Zyl-Smit et al. showed that this occurred in all three of the three studies included in the assessment, with a range of 16 80%. 27 Despite outstanding questions on the interpretation of QFT test results, the use of these tests was seen as cost-effective in a targeted screening intervention in individuals with a high risk of HIV infection in Mexico. Burgos et al. used a Markov Chain Monte Carlo modelling approach to assess the costs per LTBI case detected, TB case averted and quality-adjusted life year gained over a period of 20 years. 28 Input data were from intervention studies in the community. 29 In a related study, Garfein et al. describe the high

3 TST and IGRA: 2009 year in review 1527 prevalence of LTBI (67% by IGRA) among injection drug users. 30 This group of study participants is difficult to recruit for studies. If done, there is a high risk of a biased sample due to self-selection. The approach in this study was to use a respondent-driven sampling strategy. In this variant chain-referral method, initial recruits are given an incentive to participate and an additional incentive when they recruit others. This two-step incentive scheme is crucial to obtain a nonbiased study sample. 31 A prerequisite is that the community from which participants need to be recruited is closely connected. It is pleasing to see that newer methodology finds its way into TB research. However, the general reader must be able to assess the validity of these methods. It is therefore disappointing to see that the article does not give any insight into the success of the sampling, such as equilibrium estimates. COMPARISON OF TUBERCULIN SKIN TESTING AND INTERFERON-GAMMA RELEASE ASSAYS In 2009, several studies published in the Journal compared an IGRA with the TST. Comparison of the T- SPOT.TB assay (Oxford Immunotec, Abingdon, UK) with the TST in a TB low-prevalence area, the United States, in 326 individuals judged to have a low risk for TB infection showed excellent agreement between the two tests. 32 Overall agreement between the two tests was 98.2% (95% confidence interval [CI] ), and of the eight individuals with a positive T- SPOT.TB, six had a negative TST result. A study in a Canadian TB clinic analysed the QFT-G after implementation as a routine test for TST-positive patients. 33 QFT-G was used for all 1446 TST-positive ( 5 mm) individuals. Only 39.6% of the TST-positive patients were QFT-G-positive. The authors concluded that in this low-incidence country implementation of the QFT-G as a secondary test for LTBI can significantly reduce the number of patients given LTBI treatment. This assumes that QFT-G has superior test characteristics. The proposed strategy would be in accordance with the United Kingdom s National Institute of Health and Clinical Excellence recommendations. 34 In another low-incidence country, the Netherlands, the QuantiFERON -TB Gold In-Tube (QFT-GIT) test and the T-SPOT.TB were used to test immunocompetent individuals who were close contacts of sputum smear-positive TB patients and were born in a high TB endemic country, with a TST of 5 mm. 35 Of 282 individuals with valid outcomes (and thus TST 5 mm), the QFT-GIT was positive in 53.9% and the T-SPOT.TB was positive in 59.6%. The agreement between the TST (cut-off value 10 mm or 15 mm) and the two IGRAs was considered to be poor. An interesting finding was that a positive IGRA correlated well with possible recent TB infection, and with exposure to TB in the country of origin. A low degree of agreement between TST results and QFT results was also reported by Neal et al. during a contact investigation. 24 In a setting with proven TB transmission among co-workers of an infective TB patient, 18/61 (29.5%) co-workers had a TST > 5 mm but a negative QFT result, while 10 (16.4%) had a TST result > 15 mm and a negative QFT result. This indicates the poor sensitivity of the QFT test. As described earlier, a negative QFT is difficult to interpret when the background prevalence of infection is assumed to be high, as in this study. This might also have been the case in the study by Kik et al., 35 as in the Netherlands it has been shown that transmission of infection is markedly concentrated in similar social groups, making the background prevalence among contacts in the Dutch study rather high. 36 Shovman et al. compared the TST to the QFT-G in 35 patients with rheumatoid arthritis (RA) in Israel. 37 In this study population, overall agreement between the two tests was 56%, 45% had a TST > 5 mm, 11.4% had a positive QFT-G and the QFT-G was indeterminate in 10 (28.6%) RA patients. Ten RA patients tested TST-positive and QFT-negative, and one tested TST-negative and QFT-positive. The authors concluded that the clinical significance of a negative QFT-G in TST-positive patients with low TB risk remains to be assessed. Lew et al. used the QFT-G test to test TST-positive individuals ( 10 mm) in a contact investigation of a TB outbreak in South Korea. 38 Only 7.6% of the 388 TST-positive individuals were QFT-G-positive. The studies summarised above show that discordance between TST and QFT-G is frequent. A study in Brazil assessed what factors were associated with discordance. 39 In their study in household contacts of pulmonary TB patients, 61/255 (23.9%) contacts had discordant results, 44 were TST-positive and QFT- G-negative and 17 were TST-negative and QFT-Gpositive. Few factors were associated with discordance. Household contacts with a chest X-ray showing an old lung scar were more likely to have a positive TST test and a negative QFT-G, whereas household contacts with a reported length of exposure to the index case of >1 month were more likely to have a negative TST test and a positive QFT-G. A study of health care workers in Portugal showed that TST-positive/ QFT-negative discordance increased with the number of BCG vaccinations, and was more frequent in nurses compared to other professions. 40 Age >60 years was associated with increased TST-negative/QFT-positive discordance. Another study assessing factors associated with discordance among the general population examined in the scope of contact tracing and health care workers routinely screened for TB in Germany also identified BCG vaccination as a factor associated with TST-positive/QFT-negative discordance: in addition to this, being foreign-born was associated with discordance. 41 Davidow discusses the fact that assessing sensitivity

4 1528 The International Journal of Tuberculosis and Lung Disease and specificity for IGRAs uses substitute surrogate stand ins, as no gold standard for LTBI is available. 42 To assess sensitivity, subjects with active TB are tested, and to assess specificity subjects with an extremely low risk of TB infection are used. Using a mathematical model of LTBI test specificity, Davidow shows that the specificity of any test for LTBI is theoretically affected by its sensitivity to active TB, and by the distribution of TB, LTBI and healthy controls in the tested population. The author provides two solutions: 1) distinguishing LTBI suspects from TB suspects to correctly analyse data collected by field studies; and 2) assessing whether different cut-off points for IGRA use in TB suspects and LTBI suspects were needed. INTERFERON-GAMMA RELEASE ASSAYS FOR DIAGNOSIS OF NTM Some non-tuberculous mycobacteria (NTM), such as Mycobacterium kansasii, M. marinum, M. szulgai, M. flavescens and M. gastri, possess the M. tuberculosisspecific antigens early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10). IGRAs may thus be able to assist in the diagnosis of NTMs other than M. avium-intracellulare complex (MAIC). Kobashi et al. evaluated the QuantiFERON TB-2G (QFT-2G) test in 214 patients with NTM disease that satisfied the diagnostic criteria of the American Thoracic Society. 43 The QFT-2G positivity rate was significantly lower in patients with MAIC disease compared to patients with diseases other than MAIC. The authors conclude that QFT-2G might be useful to differentiate TB from MAIC diseases. For diagnosing diseases other than MAIC, the best cutoff level for QFT-2G should be determined. ALTERNATIVE MARKERS FOR TUBERCULOSIS INFECTION Amidst the large number of studies reporting interferon-gamma (IFN-γ) as a marker for TB infection, attempts are being made to find alternative markers, among which the chemokine induced protein 10 (IP-10) has generated the most interest Lighter et al. describe a study from a setting with low prevalence of LTBI in which IP-10 was assessed as a marker for LTBI in children. 47 It showed that IP-10 production was not age-dependent, which gives it an advantage over IFN-γ in children. IP-10 production correlated well with the magnitude of exposure to TB, as was shown earlier. 48,49 As mentioned also by Ruhwald et al., a combination of an IGRA and assessment of IP-10 is likely to be more informative than either of these tests alone. 44 CONCLUSION The diagnosis of LTBI remains challenging without an objective gold standard. The well-known disadvantages of the conventional TST are just partly overcome by the newer IGRAs, resulting in a generally better sensitivity and specificity. However, the results of these tests should be interpreted with caution and take into account the expected prevalence of disease, and the possibilities of conversion and reversion of qualitative test results in serial testing. There is an urgent need for prospective studies that are welld esigned, both in size and duration, to assess the relationship between a positive IGRA test and the risk of developing active disease. The search for new diagnostics based on alternative markers of infection should continue. References 1 Styblo K. The relationship between the risk of tuberculous infection and the risk of developing infectious tuberculosis. Bull Int Union Tuberc 1985; 60: van Leth F, van der Werf M J, Borgdorff M W. Prevalence of tuberculous infection and incidence of tuberculosis: a rea ssessment of the Styblo rule. 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