According to the World health Organization, one-third of the world's population harbours Mycobacterium tuberculosis (MTB) in an asymptomatic, latent form (latent tuberculosis [TB] infection [LTBI]) but retains a lifelong risk of future disease. The control and elimination process of the global TB epidemic could be enhanced by identification and treatment of individuals with LTBI, in particular of individuals who recently acquired the infection as the risk of developing active disease is higher in the first 2 years after exposure 12.

Until recently, the tuberculin skin test (TST) has been the only tool used to detect LTBI, but this test is flawed operationally and in terms of specificity and sensitivity 3. Lately, in vitro assays have been made available that detect interferon (IFN)-gamma responses to a combination of antigens (early secreted antigenic target 6 [ESAT-6] and culture filtrate protein 10 [CFP-10]) encoded by the RD1 (region of difference) genomic segment, which is absent from most non-pathogenic mycobacteria, including Bacillus Calmette Guerin (BCG) 345. Available evidence suggests that these tests may be more accurate than TST in the diagnosis of MTB infection, either latent or active, and two tests have been approved for the diagnosis of TB 678910111213. Moreover, results of our previous studies suggest that IFN-gamma response to multiepitopic peptides from ESAT-6 and CFP-10 (RD1) proteins selected by computational analysis is associated with active TB disease 1415161718.

Several unresolved issues remain on the potential clinical use of IFN-gamma release assays 67, and one area of controversy is whether these immune assays can be used for monitoring the response to TB treatment. Conflicting results are available on the effect of therapy for active TB disease on RD1 responses with reduced or increased responses during treatment 15192021. The existing data on the effect of LTBI treatment on T-cell responses are also contradictory 22232425. One study shows declining responses 22, whereas others have shown unchanging 23, fluctuating or increasing responses during therapy 2425. It is plausible that variations in technical aspects of assay performance such as antigens used (proteins vs. peptides), assay formats (ELISA vs. ELISPOT), time of observation (during therapy or after therapy completion), and population studied (recent versus old contacts) might explain some of these discrepancies.

Consequently, we reasoned that in an attempt to find surrogate markers of the effect of LTBI therapy, it would be important to determine in a prospective study whether, in a defined population of recent healthy close contacts of patients with pulmonary TB, isoniazid (INH) for LTBI treatment could cause changes in MTB-specific immune responses to a variety of RD1 antigens, such as the RD1 overlapping peptides of the QuantiFERON TB-Gold assay (QTF-G), RD1 intact proteins and selected peptides. Moreover to evaluate whether or not a previous infection could influence such response we included a control group composed of subjects with a known past exposure to MTB.

We prospectively recruited 238 contacts of patients with sputum smear positive pulmonary TB. Among them, 146 resulted TST⁺ and 44 of them accepted to be followed over time and were included in the analysis. The study group was divided into those who did not report having had any previous exposure to a smear positive TB case (30), and those who did (14). Among the former group, 6 patients did not receive therapy on the grounds that two were contacts of a patient with multidrug-resistant TB, two had chronic hepatitis C and two refused treatment; five subjects belonging to the latter group decided to not undergo INH therapy (figure 1).

Characteristics of individuals enrolled, divided according to previous TB exposure and LTBI treatment are shown in table 1. The group of those without a previous exposure under INH therapy had a significantly younger age (p < 0.001) compared to the other groups. No significant differences among the four groups were observed for the characteristics considered.

In this pilot study, we show that INH preventive therapy is associated with a significant decrease of the in vitro RD1 responses in individuals with LTBI with a recent exposure to MTB, who did not report any exposure in the past. This decrease was observed, although to a different extent, with all RD1 antigen reagents used. It is important to note that the trend of decrease of the test using RD1 selected peptides was particularly marked after 1–2 months of therapy and that the response of the majority became undetectable after six months, while the response to the other RD1 products (overlapping peptides of the QTF-G and RD1 intact proteins) decreased more slowly and fewer negative results were observed after six months of therapy. These differences may be related to the amount and the composition of epitopes covered by the peptides and intact proteins. For example, the peptides employed in the QTF-G cover the whole CFP-10 and ESAT-6 intact proteins (in addition to having a peptide from TB7.7) 12 whereas the peptides used in our assay are few and selected in order to be highly immunogenic 14. Based on our data, this oligoclonal response appears to be a sensitive tool to monitor MTB replication as well as active TB disease, more so than the polyclonal one against all RD1 epitopes. It is also important to note that among all these recent contacts nine did not respond at the first time of observation to RD1 selected peptides. Five of them did not respond to QTF-G either, and therefore it is likely that these subjects were not infected with MTB, although they were TST⁺. Further investigations are needed to clarify the meaning of these differences. In contrast, no significant variation was observed in individuals with LTBI who, in addition to a recent exposure, had also a past exposure to contagious TB patients. Altogether, these data suggest that RD1-based assays may be a tool to monitor therapy-related changes of MTB-specific immune responses not only in patients with active disease, as previously demonstrated by us and others 15192021, but also in individuals with a recently acquired LTBI.

A few studies have been previously published on the effect of LTBI therapy on T-cell responses. Decreasing responses to CFP-10 overlapping peptides, and not to ESAT-6, were found after INH therapy in recently exposed contacts 22 that are comparable to the group of recent contacts evaluated in the present study. However, we did not find differences in the responses to the reagents CFP-10 and ESAT-6 evaluated separately (either proteins or peptides, data not shown). On the other hand, increasing and/or fluctuating IFN-gamma responses were found by Ewer et al. in children exposed for over 9 months to a case of pulmonary TB that underwent therapy (INH and rifampicin) almost a year after exposure 25. Given the continuous exposure over a considerable period of time described in that paper, it is plausible that LTBI was already established at the time of treatment, with the consequence of a different modulation of T-cell responses compared to our study in which treatment was rapidly started in those without a past contact with MTB. In addition, in this study INH was used as chemotherapy, whereas other studies reported INH plus rifampicin 25 and it is unknown if different chemotherapies may have a different impact on MTB antigen exposure to the immune system 28. Conversely Pai et al. found unchanging IFN-gamma responses to QTF-G after INH therapy in a population of health care workers (HCW) in India 23. These subjects, given their continuous exposure to MTB, might have an immune response similar to that of our group of re-infected individuals in which no change of RD1 responses was observed over time, and therefore these results are comparable to ours. These data may indicate that the conflicting results found in the literature may be due, at least in part, to the population selected (recent infection vs. past infection with re-exposure).

The reasons for the different effect of therapy in modulating RD1 responses in recently infected patients vs. those potentially reinfected, observed in the present study, are currently unclear. In several models of infectious diseases it has been shown that the immune response is strictly dependent on pathogen replication and antigenic load 293031. In studies on in vitro IFN-gamma response to TB antigens in particular, it has been hypothesized that short incubation assays (as those used in this study) detect responses of partially activated effector T cells that have recently encountered antigens in vivo, and can therefore rapidly release IFN-gamma when stimulated in vitro. This suggests a correlation between the measure of this response and antigen load 61112. All the contacts who received INH in this study were exposed to an index TB case who was infected by an INH-sensitive strain, and all these contacts had good adherence to treatment as assessed by interview. Therefore it can be hypothesized that subjects treated early after recent infection may have had a rapidly controlled infection owing to the combination of an effective treatment and an efficient immune response, with a consequent decrease of the RD1-specific effector cells. In contrast, it is likely that contacts with previous untreated LTBI host different populations of mycobacteria, characterised by different growth rates: those originating from the recent infection, which are actively replicating and on which INH is effective, and those in a dormant state that INH is not effective in killing 28. This pool of dormant bacilli may be the cause of the longer persistence of the T cell response over time and also be responsible for the generation and maintenance of a large population of memory cells, similar to that obtained after several boosts of vaccination; at the time of the present study, reinfection may have caused a rapid and strong effector/memory response caused by the expansion of this pool of central memory cells 32. However it is possible that this response would decrease at later time points than the 6 months of our study. Therefore, future investigations are needed to evaluate the role of effector and memory T cells on the modulation of this long lasting response not only within, but also beyond the period of treatment. Based on all these observations, although LTBI has been proposed to be a static process 33, our data favour a dynamic model of LTBI, whereby subpopulations of actively replicating bacilli are controlled by the immune response 1822232425.

Some potential limitations of the present study should be considered. Firstly, we were able to document only 9 cuticonversions among the 24 individuals who did not report a previous exposure to MTB. This limit however is common to previous studies 2225, and it is important to note that the risk of previous unknown exposures in these individuals is very low because the subjects enrolled were young (median age 21 years) and the majority were born in Italy (68%) where the incidence of TB is low (8 per 100,000 inhabitants in 2004, including 39% of foreign-born individuals) 34. Secondly, TB exposure was assessed by administering a questionnaire, and thus this information may be affected by recall bias. Thirdly, INH was not administered as directly observed therapy and consequently the evaluation of adherence, performed by patient interview, may have been imprecise. Lastly, generalisation of our results may be limited by the fact that a relatively low number of TST+ subjects were available for follow-up, since many individuals refused further blood drawing.

In conclusion, our preliminary data suggest the possibility of using RD1 immune responses as surrogate markers of efficacy during LTBI treatment. A larger study is needed to better evaluate the difference in kinetics of the T-cell response after exposure to MTB in those with an already established LTBI vs. those recently infected.

• BCG: Bacillus Calmette Guerin

• CFP: culture filtrate protein

• ESAT: early secreted antigenic target

• HIV: Human Immunodeficiency Virus infection

• IFN: interferon

• INH: isoniazid

• IU: international unit

• LTBI: Latent tuberculosis infection

• MTB: Mycobacterium tuberculosis

• PHA: Phytohemagglutinin

• PPD: purified protein derivative

• QFT-G: QuantiFERON TB Gold

• RD: region of difference

• RD1: CFP-10 and ESAT-6

• TST: tuberculin skin test

• TB: Tuberculosis

• WBE: whole blood ELISA

DG, RC, FP and EG have a patent pending on T cell assay based on RD1 selected peptides.

DG designed the study and recruited the adult patients and performed data analysis and wrote the draft of the manuscript, MPP carried out the data base of collected data and helped in the data and statistical analysis, OB and FB and RC carried out the immunological assays, DD and GA helped in the recruitment of adults and children respectively and in the design of the study, CN helped in writing the draft and did the editing and performed data analysis, GI and FP helped in writing the draft of the paper and in the design of the study, EG conceived of the study and participated in its design and performed the statistical analysis and helped to draft the manuscript. The article has not been submitted elsewhere and all co-authors have read and approved the final manuscript with its conclusions.