Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). There are an estimated 8 million new cases and 2 million deaths from TB annually. Uganda is one of the world’s 22 highest burden countries with TB, with an annual risk of infection of 3% and annual incidence of new smear positive TB cases of 9.2 per 1000 in an urban setting [1].

Transmission of Mtb requires repeated close contact with someone who has active pulmonary TB and the likelihood of transmission increases with increasing levels of contact. Therefore, it is not surprising that markers of close contact, such as crowded quarters, urban living, and lower socio-economic status, are associated with acquisition of Mtb [2, 3]. Clinical characteristics of the person with pulmonary TB that increase the risk of contacts becoming infected include cavitary disease, sputum smear grade, and extent of disease based on x-ray [4–8].

The pathogenesis of TB follows a two-stage process [8]. Infection is the first stage, where, Mtb infection is contained but not eliminated by the innate and adaptive immune response. The second stage consists of progression from infection to active disease, and presents with signs and symptoms caused by the increasing mycobacterial load and the host response; 5-10% of individuals infected with Mtb develop clinical disease. Interestingly, a small percentage of individuals in TB-endemic settings never demonstrate evidence of latent Mtb infection despite persistent and repeated exposure. These individuals consistently test negative by the tuberculin skin test (TST) and/or interferon-γ response assay [9]. Persistently TST negative (PTST-) individuals may be (relatively) resistant to Mtb infection, and little is known about them [10]. Previous population-based and case-contact studies have identified epidemiological risk factors for infection [2, 11] but have not characterized PTST- individuals specifically. A few studies in healthcare settings have identified PTST- individuals [12–14] but these studies were limited in their duration of follow-up and sample size, and because they were conducted in health care settings, not generalizable to a population-based setting. Though we have examined immunological and genetic correlates of the PTST- phenotype [9, 15], these individuals have not been thoroughly characterized epidemiologically. This study examines well-characterized risk factors for Mtb infection to determine if specific epidemiologic factors explain the PTST- phenotype, and to assess whether they are simply less likely to acquire Mtb infection.

Data were collected on 1318 individuals from 454 households from April 2002 to January 2008, in which 1210 household contacts had at least one year (12 months) of TSTs in individuals who were TST negative at baseline and did not develop culture-confirmed TB during two years of follow-up; 900 (74.4%) were TST + at baseline, 168 (13.8%) converted their TST, and 142 (11.7%) were persistently TST- (PTST-). The remaining 108 individuals were TST- at baseline but did not have follow-up TSTs (TST follow-up rate of 91.8%), and thus were excluded from this analysis. When individuals that were lost to follow-up were compared to those in the analysis dataset, the only difference was in the 5–15 age group (p = 0.008), where 11.9% were lost to follow-up, compared to less than 10% for the other age groups (data not shown).

Risk score

Next, we examined a risk score (Figure 1) [11], that was evaluated differently for children less than 15 years old (Additional file 1: Table S3) versus adults 15 years and older (Additional file 1: Table S4). Because our earlier analyses (Table 1) showed that age was a significant predictor of TST status and thus a confounder, we stratified by age group at the outset. This risk score demonstrated that all the children were highly exposed, with 95% having scores of 5 and above (Table 5). Furthermore, we observed no significant difference in the distribution of scores between the 3 clinical groups for the 0–2 and 2–5 age groups. In the 5–15 age group only, there were significant differences across the three groups (P = 0.009). In adults, there was no significant difference between the clinical groups in the distribution of the scores. Adults had a slightly lower degree of risk, with the percentage of individuals with scores of 6 and above ranging between 74.2% in PTST- to 90.8% in TSTC, though this difference was not statistically significant (Table 5 and Additional file 1: Table S4). When analyzing the risk score as a quantitative variable (Figure 1), the same conclusions can be drawn, with significant differences only occurring in the 5–15 age group (P = 0.006).

	PTST-	TSTC	TST+
2 years or less	19/19 (100%)	21/22 (95.5%)	101/103 (98.1%)
>2 and equal to 5	27/27 (100%)	24/24 (100%)	93/93 (100%)
>5 or less than 15	62/65 (95.4%)	45/47 (95.7%)	231/237 (97.5%)
>15	23/31 (74.2%)	68/75 (90.7%)	410/467 (87.8%)

“High risk” is defined as a pediatric risk score ≥5 out of 9, or an adult risk score ≥6 out of 10. Components of the risk score can be found in the Additional file 1, No statistically significant differences seen across clinical groups within age group (p > 0.05).

PTST- = Persistently negative tuberculin skin test; TSTC = Tuberculin skin test converter; TST + = tuberculin skin test positive at baseline.

Then, we stratified the adults into high and low risk groups according to this risk score, and re-analyzed the variables that were significant in our earlier analyses in Tables 2 and 3 but were not part of the risk score. Results are shown in Table 6. In the high-risk group, there were no longer differences between PTST- individuals and TSTC and TST+. Interestingly, this analysis demonstrated that the variables that were significant in the ordinal regression were no longer significant after accounting for an accumulation of other factors. Differences between individuals that were TST negative versus positive at baseline persisted (P < 0.0005, Table 6).

	PTST-	TSTC	TST+	Comparison across all 3 groups	Comparison of PTST- vs TSTC + TST+	Comparison of PTST- and TSTC vs TST+
Characteristics of household
Number of windows in house
Mean (SD)	3.48 (3.25)	3.10 (2.78)	1.93 (2.03)	< 0.0005	0.056	< 0.0005
Number of rooms in house
Mean (SD)	4.26 (4.06)	3.87 (3.23)	2.77 (2.42)	0.002	0.096	< 0.0005
# People in household/room
Mean (SD)	8.22 (7.30)	7.30 (5.88)	5.99 (3.93)	0.198	0.259	0.030
Characteristics of index case
Presence of cavitary disease
No	12 (54.5%)	28 (42.4%)	155 (38.4%)
Yes	10 (45.5%)	38 (57.6%)	249 (61.6%)	0.290	0.181	0.231
Baseline extent of disease on chest x-ray
Normal	3 (13.6%)	5 (7.6%)	13 (3.2%)
Minimal	5 (22.7%)	7 (10.6%)	44 (10.9%)
Moderate	7 (31.8%)	29 (43.9%)	145 (35.9%)
Advanced	7 (31.8%)	25 (37.9%)	202 (50.0%)	0.069	0.085	0.033

“High risk” defined as a score ≥ 6 out of 10 on risk score (based on Mandalakas et al. criteria).

PTST- = Persistently negative tuberculin skin test; TSTC = Tuberculin skin test converter; TST + = tuberculin skin test positive at baseline.

Although many studies have evaluated risk factors for Mtb infection, few have looked at PTST- individuals. This large longitudinal study provides a unique opportunity to consider epidemiological factors associated with relative resistance to Mtb infection. Our study assessed household factors, individual factors, and index case characteristics that have been previously associated with presence of Mtb infection [2, 4, 5, 11]. Several factors related to exposure intensity showed increasing prevalence from PTST- to TSTC to TST+. However, these risk factors do not exist in isolation, so the examination of a risk score such as that developed by Mandalakas et al. [11] may be more appropriate, as it reflects an accumulation of risk factors. We observed no significant differences in risk score distribution by TST status except in the 5–15 age group. Thus, when considering epidemiologic risk as an accumulation of risk factors, PTST- is not determined by decreased epidemiologic risk in young children and adults.

The 5–15 age group is the only group where differences were seen in the epidemiologic risk score between PTST-, TSTC and TST+. There are several possible explanations for this observation. First, children in this age group start attending school, and thus may start to have exposure to TB cases outside the home. Second, we have previously observed a significant difference in Mtb-specific interferon-γ responses in 5–15 year olds compared to younger children [22], reflecting the so-called “golden age of immunity”. The risk of progression from infection to disease is also lowest in the 5–10 year range [24], further reflecting a developing immune response.

One of the major characteristics of the PTST- group is a significantly younger age distribution that likely reflects less cumulative exposure to Mtb. Though sharing a bed was significantly different in the three clinical groups, this is likely because the youngest individuals are PTST-, and children are most likely to share a bed with their parent. When analyses were stratified by age group, factors such as sharing meals with the index case, severity of disease in the index case, crowding, and poor ventilation, that were previously statistically significant no longer were associated (data not shown). TST induration at baseline was also significantly different between PTST- and TSTC [15, 25], suggesting that some individuals who were TST- at enrollment were progressing toward TST conversion, or perhaps that standard TST cutoff values resulted in misclassification.

A limitation of this analysis is that we did not conduct interferon-γ response assays (IGRA), on these study subjects to verify Mtb infection status. Many of these study participants were enrolled prior to the availability of the highly Mtb-specific IGRA. IGRAs were designed to avoid concerns of cross-reactivity with BCG and to allay concerns about boosting effects after repeated TSTs [19, 26, 27]. Indeed, our previous work shows that TSTC have a higher whole blood interferon-γ response at baseline than PTST- [15], so it is possible that an IGRA would have distinguished these two groups at baseline. However, the sensitivity [26, 27] and cost-effectiveness [28] of IGRAs in TB-endemic settings is still a topic of debate. In addition, the risk score developed by Mandalakas et al. [11] can be used as a proxy for assessing infection, and the authors propose their risk score could be used in lieu of the TST or IGRA. However, this risk score has not been validated in independent studies, and in fact, our results suggest that the score does not associate with latent Mtb infection as was its developed purpose. In addition, the population studied by Mandalakas et al. was restricted to HIV negative individuals. Though our analyses included HIV positive individuals, their influence on the analysis was minimal, as only 1.7% of 0–2 year olds and 2.6% of 2–5 year olds were HIV positive. Furthermore, studies have shown that IGRA positivity is associated with indicators of recent and close Mtb exposure [26, 27], which we have captured with the risk score. Another limitation is that there may be unmeasured exposure factors that could characterize PTST- individuals. We believe this is unlikely because our data collection was extensive.

In summary, we examined a number of well-established variables associated with Mtb infection and disease, and found that a risk score consisting of factors associated with transmission of Mtb did not distinguish PTST- individuals from those who eventually convert their TST or were TST positive at baseline. There are likely host factors, such as host genetics [9] and immune response [15] that explain why these individuals remain relatively resistant to Mtb infection. Alternatively, unmeasured components of shared environment, such as nutrition [29, 30] or quantity of Mtb bacilli in the air [31–33], may potentially explain why some individuals remain PTST-. Further study is needed to examine the combined influences of genetics, immunology, and nutrition on resistance to Mtb infection, as well as explore additional epidemiological factors.