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T Cell–Based Tracking of Multidrug Resistant Tuberculosis Infection after Brief Exposure

Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital; Centre for Statistics in Medicine, Institute of Health Sciences, Oxford, United Kingdom; Respiratory Disease and Pediatric Clinics, University of Modena and Reggio Emilia; and Azienda Ospedaliera Policlinico di Modena, Modena, Italy

	ABSTRACT

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Molecular epidemiology indicates significant transmission of Mycobacterium tuberculosis after casual contact with infectious tuberculosis cases. We investigated M. tuberculosis transmission after brief exposure using a T cell–based assay, the enzyme-linked-immunospot (ELISPOT) for IFN-. After childbirth, a mother was diagnosed with sputum smear-positive multidrug-resistant tuberculosis. Forty-one neonates and 47 adults were present during her admission on the maternity unit; 11 weeks later, all underwent tuberculin skin testing (TST) and ELISPOT. We correlated test results with markers of exposure to the index case. The participants, who were asymptomatic and predominantly had no prior tuberculosis exposure, had 6.05 hours mean exposure (range: 0–65 hours) to the index case. Seventeen individuals, including two newborns, were ELISPOT-positive, and ELISPOT results correlated significantly with three of four predefined measures of tuberculosis exposure. For each hour sharing room air with the index case, the odds of a positive ELISPOT result increased by 1.05 (95% CI: 1.02–1.09, p = 0.003). Only four adults were TST-positive and TST results did not correlate with exposure. Thus, ELISPOT, but not TST, suggested quite extensive nosocomial transmission of multidrug-resistant M. tuberculosis after brief exposure. These results help to explain the apparent importance of casual contact for tuberculosis transmission, and may have implications for prevention.

Key Words: contact tracing • nosocomial • T cell • tuberculin skin test • tuberculosis

Large studies of tuberculosis (TB) contacts have shown that airborne transmission of Mycobacterium tuberculosis is promoted by prolonged and close contact with an infectious case and the key determinant is the amount of time spent sharing room air with the index case (1, 2). However, mounting molecular epidemiologic evidence, using several independent strain typing methods, indicates that a significant proportion of active TB cases must have acquired M. tuberculosis infection from casual contact, often involving only short periods of time in the same room (3–10). Hence, a better understanding of M. tuberculosis transmission after brief exposure to infectious cases could help improve TB control. However, most new infections result in latent TB infection not active disease, and are therefore not amenable to molecular strain typing (11). Previous studies have relied exclusively on the tuberculin skin test (TST), which, until recently, was the only available means for studying the transmission of M. tuberculosis from an infectious source case to asymptomatic contacts (12). It remains unclear, how much exposure to an infectious case constitutes a significant risk of transmission, although some guidelines—based on TST conversions after short, defined exposures to infectious TB cases in closed environments (13)—suggest that the risk of transmission is insignificant below 8 hours (14).

The recent advent of a rapid technique for detecting antigen-specific T cells direct from blood offers a new approach for detecting M. tuberculosis infection (15–17). The ex vivo enzyme-linked immunospot (ELISPOT) assay for IFN- detects Th1-type T cells specific for ESAT-6 and CFP10 (18), antigens that are present in M. tuberculosis but absent from Bacile Calmette-Guérin (BCG) and most environmental mycobacteria (19, 20). The assay is a sensitive and specific marker of M. tuberculosis infection, gives uniformly negative results in healthy unexposed controls, and correlates better with M. tuberculosis exposure among close contacts than TST (21, 22). Hence, it might help to improve our understanding of M. tuberculosis transmission and the biology of early M. tuberculosis infection following brief exposure to infectious cases of TB.

We used ELISPOT in parallel with TST to investigate a not uncommon clinical scenario of public health importance: nosocomial exposure to an infectious case of multidrug-resistant (MDR) TB. A 24-year-old Moldovan female illegal immigrant, resident in Italy, was diagnosed with sputum smear–positive cavitary MDR TB 1 week after she had given birth to a healthy baby on the maternity unit of the University Hospital of Modena, Italy. As the highly infectious index case had been present on the maternity unit for 4 days, this posed a serious health risk to other mothers and relatives on the unit, and particularly to neonates in their first week of life who were delivered on the unit during the index case's stay. Analysis of hospital records identified 41 babies and 47 adults who were present on the maternity unit for varying amounts of time during the index case's admission. These records also enabled accurate estimates of the amount of time each contact spent sharing room air with the index case. We screened 88 hospital contacts of this highly infectious index case by ELISPOT and TST 11 weeks after the point-source exposure, as well as four household contacts. Given that TST and ELISPOT measure different immunologic responses to different stimuli, we included purified protein derivative (PPD) in our ELISPOT assays to help differentiate these differences. This allowed us to compare the in vivo delayed-type hypersensitivity (DTH) response to PPD with the ex vivo Th1-type antigen-specific T cell response to tuberculin PPD, as well as to ESAT-6 and CFP10.

Our study was designed to answer the following three questions. First, how well do ELISPOT and TST results correlate with exposure to MDR M. tuberculosis when the duration of contact is relatively short? Second, how extensive is transmission of MDR M. tuberculosis in this nosocomial setting? Third, given that neonates have severely impaired DTH responses to M. tuberculosis, is it possible to detect antigen-specific T cell responses after M. tuberculosis exposure in the first week of life? Some of the results of these studies have been previously reported in the form of abstracts (23, 24).

	METHODS

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Participants
Ninety-two individuals were identified as having been present on the maternity unit during the time that the index case was admitted. Of these, 88 had only nosocomial contact, whereas the other four had both household and nosocomial contact. Forty-one were newborn babies, 42 were mothers, and nine were visiting relatives (four husbands and five grandparents). All adult contacts were informed by mail and/or telephone of their potential exposure to MDR TB and were invited to participate in the study. The study was approved by the local ethics committee, and each study participant provided written informed consent. For infants, both parents were required to sign the informed consent form.

Study Protocol
There is little published guidance for the management of contacts of MDR TB (25), and there are no European or Italian guidelines; in particular, the effectiveness of preventive therapy with second-line agents for contacts of MDR TB is unknown. Our study protocol, which was developed in consultation with international experts and approved by the Modena Ethics Committee, stipulated that participants with a positive TST or ELISPOT result would undergo chest radiography and chest high resolution computed tomography. If either of these were abnormal, contacts would have a complete clinical investigation for active TB. Participants who were positive by either test but with normal radiologic test results and no symptoms, would receive enhanced clinical and radiologic surveillance for 2 years, so as to allow early detection and treatment of incipient active MDR TB.

Procedures
A detailed standardized questionnaire was administered to all adult participants by two chest physicians (LR and PR), which elicited the following: age; ethnicity; place of birth; BCG vaccination history; risk factors for previous TB exposure; movement on the unit during the admission period of the index case; whether they recognized the index case (based on verbal descriptions of her physical appearance, and the bedroom and bed number in which she stayed); and whether they recalled one or more conversations with the index case. All participants underwent venepuncture and TST over a 5-day period that began 11 weeks after the index case had left the hospital. Blood samples (10 ml from adults and 2 ml from newborns) were drawn into heparinized tubes immediately before TSTs were placed, and ELISPOT assays were performed within 2–4 hours. TSTs were administered by the Mantoux method using 5 IU (0.1 ml) of PPD-Siebert (PPD-S), (Biocine Test PPD; Sclavo, Siena, Italy), as stipulated in international guidelines (12). TSTs were performed and read by two experienced pulmonologists (LR and PR) who routinely look after tuberculosis patients and who were blinded to the ELISPOT results. The cutaneous appearance of peau d'orange was noted in all participants, confirming intradermal inoculation of PPD. Induration was measured after 72 hours with a ruler. TST response was scored as positive if induration diameter was equal to or greater than 5 mm (irrespective of BCG vaccination status), in accordance with international guidelines for recent contacts (12). Documentation of BCG vaccination scars was also carried out by the same two pulmonologists.

Ascertainment of Exposure to the Index Case
Mothers were admitted to one of three six-bedded rooms or one of two three-bedded rooms on the maternity unit; roommates slept in the same room overnight, shared the same table for meals, and the same lavatory. The index case was admitted in late November when windows and doors were almost always shut and no air conditioning or ducted ventilation were present. Mothers had their babies close to their beds during the daytime, whereas all babies slept in the nursery without their mothers at night. Other sites of potential contact with the index case were not quantifiable and comprised common spaces, such as corridors. The possibility of significant contact in these areas could not be excluded, although the questionnaire did record those mothers who could recall one or more conversations with the index case.

Exposure to the index case was estimated for the hospital contacts (excluding the household contacts) by comparing their time of stay on the maternity unit and case notes with those of the index case and by using relevant information from the questionnaires. The following measures of exposure were recorded: (1) whether or not admitted in the same bedroom as the index case (which was one of the three six-bedded rooms, designated Room 3); (2) total time spent sharing room air with the index case (in all rooms on the maternity unit, including Room 3); (3) knowledge of the index case; and (4) reporting at least one conversation with the index case. The exposure of the four household contacts to the index case was much greater than that of the nosocomial contacts but could not be accurately quantified; they were therefore classified as a separate group. However, this group was included in a second analysis (comprising the 88 nosocomial contacts and the four household contacts) based on four broad categories of increasing exposure to M. tuberculosis, stratified by proximity to the index case. Participants were assigned to these categories as follows: individuals admitted to the maternity unit at the same time as the index case but with no discernible contact with her based upon hospital records, case notes, and questionnaires; individuals with direct exposure to the index case based upon hospital records, case notes, and questionnaires but who did not sleep in room three; mothers who slept in Room 3 during the hospital stay of the index case; and household contacts.

Ex vivo ELISPOT Assays
Assays were performed as previously described using 35 overlapping peptides spanning the length of ESAT-6 and CFP10 (15, 22, 26, 27), rESAT-6 (a kind gift of A. Whalen, VLA, Weybridge, UK), rCFP10 (a kind gift of M. Singh, Braunschweig, Germany), PPD (RT-49; Statens Serum Institut, Copenhagen, Denmark), and phytohaemagglutinin (ICN Biomedicals, Costa Mesa, CA) as positive control. rESAT-6 and rCFP10 were expressed in, and purified from, recombinant Escherichia coli; purity and absence of residual contaminating E. coli proteins were confirmed by SDS-PAGE followed by silver staining (rESAT-6) or Coomassie blue staining (rCFP10) and Western blotting using polyclonal anti–E. coli antibodies (28). Peptides were grouped in 12 pools, as previously described (22). All test conditions were carried out in duplicate wells and pairs of wells were scored positive if they contained a mean of at least five spot-forming cells more than the mean of the negative control wells, and, in addition, this number was at least twice the mean of that for the negative control wells (16, 22). The cut-off point for recombinant antigens was 10 spot-forming cells above background, as previously established for this preparation of antigen (21, 27). These cut-off points were defined before unblinding the results. Assays were deemed positive if there was a positive response to one or more pools of the ESAT-6– or CFP10-derived peptides, or to rESAT-6 or rCFP10.

Dry plates were scored using an automated ELISPOT counter (AID-GmbH, Strassberg, Germany). Intensity and spot size settings were predefined using several plates that were clearly negative or positive, and those same settings were used throughout.

Statistical Methods
All analyses and statistical tests used were defined prior to unblinding. The first analysis focused on estimating the strength of association between exposure to the index case and the RD1 ELISPOT, PPD ELISPOT, and TST results. Estimates of the relative increase in the odds of a positive test result for each hour of time sharing room air with the index case, and for each of the other three markers of exposure to the index case (sleeping in Room 3; having a conversation with her; and recalling her identity) across the 88 nosocomial contacts were obtained from logistic regression. The second analysis was based on the four categories of exposure including the four household contacts, and estimated the average relative increase in odds between consecutive categories of exposure. In addition, analyses were undertaken to investigate whether ELISPOT and TST results were associated with BCG vaccination and place of birth. Analyses were performed using STATA (Stata Corporation, College Station, TX).

	RESULTS

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Demographic and Clinical Characteristics of Study Participants
TST, RD1 ELISPOT, and PPD ELISPOT results and demographic characteristics for all 88 hospital contacts and the four household contacts are shown in Table 1 . The majority of contacts were Italian-born white individuals with very low risk for prior M. tuberculosis exposure and had not been BCG-vaccinated. All mothers had been tested for HIV antibodies and were HIV seronegative. The mean duration of exposure to the index case for the 88 potential hospital contacts was 6.05 hours (range: 0–65 hours), with 15 contacts having more than 8 hours exposure, and 63 with no quantifiable direct exposure. For the four household contacts, the duration of exposure to the index case was much greater but could not be precisely quantified. The three adult household contacts were exposed during the last 4 months of pregnancy of the index case when she had a productive cough, and her baby had been at home with her for a week before she was diagnosed and put in respiratory isolation at the hospital. Only one contact, the baby of the index case, had received prophylactic chemotherapy at the time of testing; pyrazinamide and ethambutol had been initiated 7 weeks previously.

Results of TST, RD1 ELISPOT, and PPD ELISPOT Assays
Results of all three assays for all 92 participants are shown in Figure 1 . Only four individuals were TST positive, whereas 17 were positive by RD1 ELISPOT and 34 by PPD ELISPOT (Figure 1). The mean number of IFN- spot-forming cells for rESAT-6 and rCFP10 combined in the 17 RD1 ELISPOT-positive contacts was 83/million periphereal blood mononuclear cells (inter-quartile range: 42–95/million periphereal blood mononuclear cells). Of the 17 RD1 ELISPOT–positive contacts, 13 had a response to one or the other antigen that was 50% or more above the threshold for a positive response. Only two had responses to peptide pools in addition to recombinant antigen. MO3, the husband of the index case, who had active MDR TB and was TST negative, responded to one pool of ESAT-6–derived peptides and one pool of CFP10-derived peptides, and MO95, who was positive by TST (10 mm induration), responded to two pools of ESAT-6–derived peptides and three pools of CFP10-derived peptides. It is surprising that so few of the 17 RD1 ELISPOT–positive contacts responded to peptide as well as to recombinant antigen; the significance of this is currently uncertain.

View larger version (14K): [in this window] [in a new window]   Figure 1. Concordance of test results for tuberculin skin testing (TST), RD1 enzyme-linked immunospot (ELISPOT), and purified protein derivate (PPD) ELISPOT for all 92 participants (88 nosocomial and 4 household contacts). Numbers within circles represent positive results and show overlap between assays. A total of 17 individuals responded by RD1 ELISPOT: 15 to rESAT-6 antigen; 12 to rCFP-10 antigen; 10 to both antigens; one to ESAT-6 peptides and rESAT-6 antigen; and one to ESAT-6 peptides, CFP10 peptides, rESAT-6 antigen, and rCFP10 antigen. A total of 34 contacts responded by PPD ELISPOT, and four responded by TST. *TST results for these two individuals were 5 and 10 mm; TST results for these two individuals were 5 and 8 mm.

RD1 ELISPOT Correlates Closely with Nosocomial Exposure to MDR M. tuberculosis
Figure 2 shows the prevalence of positive test results for each assay among the four predefined broad groups of contacts, stratified by overall proximity to the index case, with the most heavily exposed contacts, the household contacts, in the center. The odds for a test result being positive for each increase across the stratified exposure groups increased by 1.93 (95% CI 1.11–3.35, p = 0.020) for RD1 ELISPOT and 1.84 (1.09–3.11, p = 0.022) for PPD ELISPOT. There was no significant correlation for TST.

View larger version (26K): [in this window] [in a new window]   Figure 2. RD1 ELISPOT, PPD ELISPOT, and TST results for all 92 participants (88 nosocomial contacts and 4 household contacts) stratified by increasing proximity to the index case. The ratio of positive contacts to total contacts assayed (and percentage) are shown in the appropriate square for each category of tuberculosis (TB) exposure. CI = confidence interval; HHC* = household contacts; OR = odds ratios of the correlation of each of the tests with intensity of M. tuberculosis exposure. The four household contacts were: the husband of the source case, who had subclinical active pulmonary multidrug resistant (MDR) TB (negative by TST, positive by RD1 ELISPOT and PPD ELISPOT); the husband's mother and father (both negative by TST and RD1 ELISPOT but positive by PPD ELISPOT); the baby of the index case who had been started on preventative therapy 7 weeks before testing (negative by all assays). Room 3 = bedroom of index case; in this group, 11 individuals knew the index case, of whom eight could recall a conversation and 10 had quantifiable direct exposure to the index case from hospital records; a further five had quantifiable direct exposure to the index case as determined from hospital records, but did not know the identity of the index case.

For the 11 contacts who could recall the identity of the index case, there was a trend toward an increase in odds of a positive RD1 ELISPOT result (Table 2). Eight of these individuals also had a conversation with the index case, and were significantly more likely to be RD1 ELISPOT–positive than contacts who had not conversed with her (Table 2). Neither PPD ELISPOT nor TST were more likely to be positive in these individuals.

Correlation Between Assay Results and BCG Vaccination
RD1 ELISPOT results were independent of BCG vaccination status and foreign birth, whereas PPD ELISPOT results were strongly associated with both of these factors (Table 2). As all four TST-positive participants were foreign born, and three were BCG vaccinated, correlation between TST results and these factors was not statistically estimable.

Agreement Between Results from Different Assays
Agreement between assay results was quite limited; only two individuals had positive results in all three assays, and 52 individuals had negative results in all three assays (Figure 1). Although 81.5% of participants had concordant results for RD1 ELISPOT and TST, the level of agreement (kappa [] value) was not significant ( = 0.138, p = 0.088). There was a low level of agreement (beyond chance) for RD1 ELISPOT and PPD ELISPOT ( = 0.247, p = 0.010) and for PPD ELISPOT and TST ( = 0.162, p = 0.005). Of the 17 RD1 ELISPOT–positive participants, two were TST-positive and PPD ELISPOT–positive, and nine were PPD ELISPOT–positive (Figure 1). Interestingly, six RD1 ELISPOT–positive individuals were negative by both TST and PPD ELISPOT, including the two RD1 ELISPOT–positive babies (Figure 1).

Analysis of Discordant Results
Because RD1 ELISPOT and TST results have previously shown a high level of agreement (22), we analyzed the concordant and discordant results for these assays. Table 3 shows that the 15 individuals who were RD1 ELISPOT–positive and TST-negative had significantly higher markers of exposure to M. tuberculosis than the 73 individuals who were negative by both tests. Thus, an isolated positive ELISPOT result was a good predictor of M. tuberculosis exposure. As only two participants were TST-positive and RD1 ELISPOT–negative, statistical comparison of this group with others in Table 3 is not possible; however, it is notable that neither individual had slept in the same room as the index case but both were foreign-born immigrants and one was BCG vaccinated.

	DISCUSSION

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Unlike the RD1 ELISPOT, the PPD ELISPOT was also strongly correlated with BCG vaccination status, which reflects the antigenic cross-reactivity of PPD with BCG. PPD ELISPOT results were also significantly more likely to be positive in foreign-born individuals, predominantly from tropical countries, a risk factor for environmental mycobacterium exposure and M. tuberculosis exposure. Given that the RD1 ELISPOT correlated strongly with all other measures of M. tuberculosis exposure, its independence from foreign birth suggests that, unlike PPD-based assays, it is not confounded by environmental mycobacterial exposure. Although foreign-born individuals were more likely to be PPD ELISPOT–positive, 22 of 38 (58%) Italian-born, unvaccinated adults were weakly PPD ELISPOT–positive. In contrast, none of the 11-week-old babies, of which there were 41, were positive by PPD ELISPOT. These findings are consistent with the concept that PPD-specific T cells in adults may be gradually induced over time by environmental exposure to antigens with cross-reactivity to PPD, for example antigens in ubiquitous mycobacteria that may be more prevalent in warm, tropical regions (30, 31). The high sensitivity of the ELISPOT assay allowed for detection of low numbers of PPD-specific T cells that may have been induced in this manner in the unvaccinated Italian-born adults, but which were absent in newborns. Of the four TST-positive individuals, all were foreign born and three were BCG vaccinated, suggesting that these DTH responses to PPD were likely induced by BCG vaccination or environmental mycobacterial exposure.

Despite the relative immaturity of the immune system in the neonatal period, the ELISPOT assay was sufficiently sensitive to detect IFN-–secreting lymphocytes in the mitogen-stimulated positive control wells for all of the 11-week-old babies. Hence, all assays gave evaluative results, although the magnitude of the IFN- response to mitogen was lower in babies than in adults. Of the 17 contacts who were RD1 ELISPOT–positive, only two were babies, and this probably reflects the fact that as a group the babies had much lower exposure than the mothers, because no babies slept in the same room as the index case. Interestingly, the mothers of these two babies were also positive by RD1 ELISPOT. In common with all the other babies, the two RD1 ELISPOT–positive babies were negative by the other assays. The fact that these babies and six adults mounted a T cell response to ESAT-6 and CFP10 in the absence of any IFN- ELISPOT response to PPD is notable. This observation suggests that in humans, as in animal models (32, 33), these antigens are important early targets of Th1-type T lymphocytes following recent M. tuberculosis infection. Interestingly, out of four reported incidents of exposure to infectious pulmonary TB on neonatal and maternity units, none of the more than 2,000 infants who underwent skin testing had a positive TST result (34–37). Given the impaired ability of neonates to mount DTH responses to PPD (38), it is encouraging that the IFN- ELISPOT assay is capable of detecting antigen-specific T cell responses to ESAT-6 and CFP10 in some neonates with recent TB exposure. Interestingly, the baby of the index case—the most heavily exposed newborn—was negative by all three assays. This may be because the baby had received preventive therapy for 7 weeks before testing and, if he had acquired infection during his first 4 weeks of life, mycobacterial replication might have been suppressed sufficiently to prevent or delay induction of a cellular immune response.

The RD1 ELISPOT results suggest that nosocomial transmission of MDR M. tuberculosis during the 4-day admission of the highly infectious index case was quite extensive. The presence of circulating T cells in blood that are specific for M. tuberculosis antigens strongly suggests that M. tuberculosis infection has occurred because T cells can only be primed to mycobacterial antigens if pathogen-derived peptides are processed and presented in the context of major histocompatibility complex molecules on the cell surface of infected macrophages in vivo. Thus, the contacts with positive RD1 ELISPOT results must have had intracellular M. tuberculosis infection to mount a T cell response to ESAT-6 or CFP10. The strong correlation of RD1 ELISPOT results with M. tuberculosis exposure in this as well as in other studies further supports this conclusion. Because only two of the 17 RD1 ELISPOT–positive contacts were TST positive, there was a high level of discordance for these two tests, such that there was no significant agreement beyond chance. This is in contrast to a recent study of 535 students exposed over a 9-month period at school to a single highly infectious case of TB, in which the level of agreement between RD1 ELISPOT and TST was high ( = 0.72, p < 0.0001) (22). The infectiousness of the source case, who had sputum smear–positive cavitary TB and a productive cough, was probably similar to that of the case reported here. The school, as with the Modena maternity unit, was an enclosed environment without ducted ventilation, and the dimensions of the school classrooms in which the students shared lessons were similar to those of the rooms occupied by the index case on the maternity unit. Interestingly, the odds ratio for a positive RD1 ELISPOT increased by a factor of 1.04 (p < 0.0001, 95% CI: 1.02–1.05) for each hour spent sharing room air with the index case in a classroom. Thus, the transmission dynamics of M. tuberculosis inferred from RD1 ELISPOT results in these two settings, which share similar key features, were almost identical.

Why then was the agreement of TST results with RD1 ELISPOT so high at the school and so low on the maternity unit? There were four pertinent differences between the two studies. First, although the M. tuberculosis strain on the maternity unit was MDR, whereas that at the school was fully drug sensitive, there is no convincing data that MDR M. tuberculosis is less likely to induce a positive TST than drug susceptible strains (39, 40). Second, although TST at the school was performed using the Heaf (22) method and not the Mantoux method, this is unlikely to account for the difference, because there is, in general, a good correlation between these two methods of TST administration, and the Mantoux is, if anything, the more sensitive of the two (12). Failure in Mantoux technique or in the tuberculin reagent seem very unlikely, as all skin testing was performed by two experienced pulmonologists who, at the same time as this study and using the same batch of PPD, regularly elicited positive TST results in patients with tuberculosis in their routine practices. Third, TST at the school were performed 3 months after the end of a 9-month period of exposure, whereas the nosocomial contacts in this study were tested 11 weeks after a 4-day exposure. Thus, the majority of students at the school were tested more than 11 weeks after their exposure, and it could be argued that there was insufficient time for the TST to convert in our nosocomial contacts. However, this is unlikely to be a significant factor, as studies have previously shown that the vast majority of people who develop a positive TST after exposure to M. tuberculosis do so within 12 weeks (12, 38, 41, 42). Thus, it is unlikely that a significant number of our cohort would become TST-positive if retested at later time points. Rather, the most significant difference between the two settings was the mean duration of exposure to M. tuberculosis, which was 62.2 hours (range 0–453 hours) at the school, much higher than the 6.05 hours (range 0–65 hours) on the maternity unit (p = 0.003, Mann-Whitney U test).

We believe that in this study, the high sensitivity of the RD1 ELISPOT has allowed us to detect extensive transmission of M. tuberculosis at relatively low levels of exposure that were insufficient to induce a positive TST. This may be because lower exposures to M. tuberculosis result in inhalation of, and infection with, lower inocula of bacilli, which are sufficient to induce a detectable T cell response to secreted immunodominant M. tuberculosis antigens by ex vivo ELISPOT, but too low to induce a cutaneous DTH response to PPD. Frequencies of ESAT-6 and CFP10-specific IFN-–secreting T cells enumerated by ELISPOT were generally low in RD1 ELISPOT–positive TST-negative contacts (mean = 82 IFN- spot forming cells/million PBMC), and a clinically measurable cutaneous DTH response may require higher numbers of Th1-type antigen-specific T cells. Indeed, the extent of cutaneous induration correlated closely with the frequency of ESAT-6 and CFP10-specific IFN-–secreting T cells based on TST and ELISPOT results of the 535 students at the school (K. Ewer and A. Lalvani, unpublished observations).

Notably, only 2 of 17 contacts who had an IFN- ELISPOT response to rESAT-6 or rCFP10 also responded to the corresponding peptides, whereas previous studies of patients with TB and TST-positive contacts with heavy TB exposure or long-standing latent TB infection exhibited cellular immune responses to peptides as well as antigens (16, 22, 27, 43). It is most unlikely that the antigen-specific responses observed here were artifacts due to contaminants, because the recombinant antigen preparations we used were of high purity (28), and we have not observed preferential responses to these antigens rather than peptides in our other studies (16, 22, 27). Rather, the unusual pattern of ELISPOT response observed might conceivably be related to the particular epidemiologic features of this investigation: recent, brief exposure to M. tuberculosis in a previously unexposed population, a setting in which ex vivo cellular immune responses to M. tuberculosis have not previously been measured. Further immunologic studies and longitudinal follow-up may help to clarify the biological significance of this pattern of response.

In conclusion, we believe that the use of RD1 ELISPOT in our epidemiologically precisely defined population, with recent, short duration, point-source exposure, made it possible for us to identify individuals who have probably been infected with a low dose of M. tuberculosis. Such a population has not been identifiable before now because previous studies had to rely solely on TST to track M. tuberculosis transmission among asymptomatic contacts. Our findings may help explain the molecular epidemiologic evidence that indicates significant M. tuberculosis transmission after brief exposure (3–5, 7–10). This raises the question of whether the RD1 ELISPOT–positive/TST-negative contacts described here have a risk of progression to active TB similar to individuals whose M. tuberculosis infection is accompanied by a positive skin test. Longitudinal follow-up of large cohorts of recent contacts with discordant test results will help to answer this clinically important question, and the participants in this study have been enrolled for such long-term surveillance.

	Acknowledgments

 
The authors thank all the parents of the newborns involved for their patience, understanding, and adherence with the surveillance protocol; John F. Murray (International Union Against Tuberculosis and Lung Disease, Paris, France), Jeffrey R. Starke (Baylor College of Medicine, Houston, TX), and Gerald H. Mazurek (Centers for Disease Control and Prevention, Atlanta, GA) for advice on study design; Mario Bertolani (Radiology Department, Azienda USL, Modena, Italy) and David M. Hansell (Department of Radiology, Royal Brompton Hospital, London, UK) for the reading of chest radiographs and HRCT scans of study participants; Maria F. Bertolani (Pediatric Clinic, University of Modena & Reggio Emilia, Modena, Italy) for her help with clinical management of newborns; Marcella De Vita and Carla Garutti (Respiratory Disease Clinic, Policlinico Hospital, Modena, Italy) for drawing of blood samples; Patrizia Marchegiano (Medical Direction, Policlinico Hospital, Modena, Italy) for coordination of logistic details related to the study; and Kerry Millington and Chris O'Callaghan (Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK) for critical appraisal of the manuscript.

	FOOTNOTES

 
Supported by grants from the Wellcome Trust, UK, and the Azienda Ospedaliera Policlinico di Modena, Italy.

L.R. and K.E. contributed equally to the study.

Correspondence and requests for reprints should be addressed to Dr. Ajit Lalvani, Nuffield Department of Clinical Medicine, University of Oxford, Level 7, John Radcliffe Hospital, Oxford OX3 9DU, UK. E-mail: ajit.lalvani{at}ndm.ox.ac.uk

Conflict of Interest Statement: L.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; K.E. is named as inventor on a pending patent relating to T cell–based diagnosis; M.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; B.M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; L.M.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; A.L. is named inventor on several patents and pending patents relating to T cell–based diagnosis filed by the University of Oxford since 1996 and regulatory approval and commercialization of ELISPOT has been undertaken by a spin-out company of the University of Oxford, Oxford Immunotec Ltd. A.L. has a share of equity in, and acts as a non-executive scientific acvisor to, Oxford Immunotec Ltd and has received $10,300 in remuneration.

Received in original form March 9, 2004; accepted in final form April 30, 2004

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