Tuberculosis is an airborne disease of poverty that mostly affects young adults in developing countries   . In 2009, WHO reported about 9.4 million new TB cases including 3.3 million women and1.1 million TB/HIV co-infected individuals  . Despite successful control of primary TB infection, some bacilli remain in a non-replicating or slowly replicating dormant state for the rest of the life of the individual  . Five to 10% of individuals with latent TB infection (LTBI) develop overt TB disease during their lifetime, mostly within 5 years of infection. The tuberculin skin test (TST) is one of the few tests that were first introduced in the 19th century  . TST introduced by Mantoux has been widely used as the screening test of choice to identify individuals with LTBI for more than a century  . TST has some limitations: low sensitivity in immune-compromised patients and a presumed cross-reactivity with hypersensitivity in BCG-vaccinated and nontuberculous mycobacteria (NTM) infections  . Recently, IFN-γ release assay (IGRA) which measures the production of IFN-γ in whole blood upon stimulation with specific mycobacterial antigens, has been introduced to diagnosed LTBI, but it is not currently routinely used   . The usefulness of TST in determining LTBI point prevalence and the sensitivity and cost-effectiveness using mycobacteria PPD for stimulation in IGRA tests have recently been demonstrated by our group   . TST and IGRA may allow triaging individuals for chemoprophylaxis to increase case detection and reduce the burden of active TB in low-resource countries  . This study aimed to determine the point prevalence of LTBI in an area with high TB prevalence in Eastern Sudan using TST and the IFN-γ release assay with PPD as stimulant.
2. Materials & Methods
2.1. Scientific and Ethical Considerations
The study was scientifically reviewed and approved by the Ethics & Scientific Committees of the Institute of Endemic Diseases, University of Khartoum and the Ethics Committees of the Federal Ministry of Health, Khartoum. Written informed consents were obtained from all participants.
2.2. Study Population
Seven hundred and sixty eight individuals [HHCs n = 523; CCs n = 245] were recruited. Data was collected using a specially designed case record form (CRF) with sections for demographic, clinical and laboratory data. Index cases with smear positive TB and at least one House Hold Contact were enrolled. HHCs were included if they were: ≥10 years old, lived for ≥ six months at the same property as the respective index case, have no previous history of anti-tuber- culous treatment and have signed an informed consent. Community Controls were enrolled if: age ≥10 years, have no history of tuberculosis in his/her household and without symptoms/signs of tuberculosis and have signed an informed consent.
2.3. Tuberculin Skin Testing (TST)
Tuberculin skin test (TST) was performed for all contacts by injecting 0.1 ml solution containing 5 tuberculin units (Razi Institute, Iran). The largest transverse diameters of indurations were measured 72 hours later using the ballpoint pen technique.
2.4. Whole Blood Stimulation and IFN-γ Release Assay
Five mls of whole blood were collected from all volunteers: three mls in lithium heparin tubes were alloquated into three tubes and stimulated with Phytoheamagglutinin (PHA) as positive control, PPD antigen (Spain diagnostics Ltd.) and no additive (negative control). Two mls in EDTA tubes were used for ESR measurement. The tubes were incubated for 24 hours at 37˚C in Cellestis incubator (Cellestis Ltd., Victoria, Australia) and the supernatant was collected after centrifugation and stored at −20˚C until assayed. IFN-γ in the supernatant was measured by ELISA (Koma Biotech, Seoul, South Korea) as per manufacturer instructions.
2.5. Statistical Analysis
Statistical analyses were performed using Epidemiological Information (Epi Info) software version 184.108.40.206. The levels of IFN-γcytokine and TST indurations between the HHCs and CCs were compared using student t-test and Chi-square tests. Pearson correlation test was used to correlate TST indurations and IFN-γ levels. P levels of <0.05 were considered significant.
3. Results (Tables 1-5)
A total of 768 consenting volunteers were recruited in the study; two hundred and forty five were HHCs for 87 index cases while five hundred and twenty three CCs were enrolled with a male: female ratio of 1:2. The mean ages of HHCs and the CCs were 35.6 ± 15.7 years and 30.6 ± 11.7 years respectively (p = 0.99). BCG vaccination as checked by the presence of scar was reported in a small percentage of volunteers (12.7%). The majority (>60%) of the study volunteers were from the indigenous tribes of Eastern Sudan [Bani Amir and Hadandawa]. The
Table 1. Baseline characteristics of the HHCs and CCs at Day 0 and Day 614 of follow up.
*Highly significant difference in Screening and follow up TST indurations (p < 0.001). **Mean IFN-γ level IU/ml significantly reduced on Day 614 of follow up.
Table 2. TST indurations, IFN-γ level and ESR in the study population at Day 0 and Day 614 of follow up.
Continuous variables are expressed as means ± SD. *Significant difference **Highly significant differences.
Table 3. TST indurations and IFN-γ production on Day 0 and Day 614 in House-Hold Contacts (HHCs) in different age groups.
*/**statistically significant differences.
Table 4. TST indurations and IFN-γ production levels on Day 0 and Day 614 in different Community Contacts (CCs) age groups.
*Statistically significant differences.
Table 5. LTBI point prevalence using TST indurations (≥10 mm) & IFN-γ production levels (≥0.35 IU/ml) in the study population at recruitment [Day 0].
overall recruitment day [D0] mean TST induration was 4.7 ± 5 mm, while it was 4.6 ± 5.5 mm and 2.8 ± 3.7for HHCs [n = 245] and CCs [n = 523] respectively (p = 0.000). The mean TST induration on Day 614 follow-up was 9.1 ± 7.2 mm for HHCs [n = 196/245; 80%] while it was 4.4 ± 3.2 for CCs [n = 272/523 (52%] (p = 0.001). TST indurations increased in HHCs in the age groups 31 - 40 and 41 - 50 compared to those in 10 - 20, 21 - 30 and ≥51 age groups on D0, with similar increase in these groups at Day 614 but with slight reduction in the ≥51 age group. Similar results were reported for CCs but with significant increases in ≥51 age group on Day 614 (p = 0.001). Using TST induration of ≥ 10 mm, the LTBI point prevalence for HHCs and CCs was 461/1000 and 367/1000 individuals respectively (p = 0.03).
The overall D0 mean IFN-γ level was 0.085 ± 0.45 IU/ml compared to 0.217 ± 0.87 IU/ml at Day 614. D0 mean IFN-γ production levels were 0.66 ± 0.17 IU/ml and 0.06 ± 0.04 for HHCs and CCs respectivelyitdropped significantly for CCs at Day 614 [HHCs 0.57 ± 0.15 IU/ml; CCs 0.02 ± 0.02; (p = 0.03). IFN-γ release was significantly high in 41 - 50 and ≥51 age groups for HHCs on D0. IFN-γ production levels [D614] dropped in the age groups 10 - 20, 21 - 30 and 31 - 40 and continued to increase in the age groups 41 - 50 and ≥51 (p = 0.001). Mean IFN-γ Levels showed significant variable levels on D0 and D614 in different age groups. Using IFN-γ of ≥0.35 IU/ml, the point prevalence was calculated for HHCs and CCs as 440/1000 and 203/1000 individuals respectively (p = 0.000000001).
The mean ESR at D0 was slightly higher for CCs compared to HHCs (p = 0.03).
Tackling the problem of Latent TB infections (LTBI) with early diagnosis and selective treatment especially among house hold contacts of patients with smear positive pulmonary can help control the disease and help reduce the risk of late presentation. TST remains the most commonly used test for LTBI diagnosis to initiate preventive treatment, this is justified by the fact that in vivo tests identified the presence of a number of cytokines and chemokines in TST reaction site; these include IL-4, IFN-γ, TNF-α, IL-10, IL-12, so TST indurations might capture an immune response in M tuberculosis-infected individuals that could be missed by the IFN-γ release assay    . Recently, Shakak and colleagues demonstrated that the reactivity of TST in duration of ≥10 mm is as good as IFN-γ release assay (IGRA) in diagnosing LTBI. In addition, it has an advantage over IFN-γ release assay since it can detect patients with progressing disease that will be missed by IGRA tests. These patients usually have evolving immune responses that predominantly exhibit Th2 cytokine patterns with secretion of large amounts of IL-10 with no or minimum IFN-γ secretion  . Our results showed that LTBI point prevalence reported by TST (induarion ≥10 mm) were not markedly different from those reported by IFN-γ release assays and were higher among HHCs compared to CCs as reported previously     . In addition, this study reported a higher LTBI point prevalence among HHCs and CCs compared to those reported from central Sudan  . This is not totally surprising, since Eastern Sudan is known to be more endemic for Mycobacterial diseases. The comparatively high LBTI point prevalence among CCs could further point to the high prevalence of TB in communities of Eastern Sudan. In this study, TST LTBI point prevalences were significantly higher than those reported by IGRA; this could probably mean that TST could be more sensitive than IGRA in picking up LTBI infections in area with high TB prevalence. The fact that females constituted the majority of the study volunteers, could be explained by the simple fact that females and children do not get equal share of nutrition and access to treatment in most of the communities in Eastern Sudan. Our results showed that increasing age is accompanied by increasing IFN-γ levels in agreement with previous studies  .
TST in duration ≥10 is a simple, cheap and accurate test for the diagnosis of LTBI especially in areas of high TB prevalence. Point prevalence of LTBI reported by TST was higher than reported by IFN-γ release assay.
The investigating team would like to thank the administrations of the department of Clinical Pathology and Immunology, Institute of Endemic Diseases, University of Khartoum for provision of logistic and financial supports.
Conflict of Interests
The authors declare that they have no conflict of interests.
SAO, EAGK conceived and wrote the study proposal, prepared the necessary scientific and ethics approvals and contributed to volunteers’ recruitment. WSES, AMM were involved in volunteers’ recruitment, care and follow-up, participated in manuscript writing and revision. All authors read and approved the final manuscript.
LTBI Latent TB Infection
HHCs House Hold Contacts
CCs Community Contacts
TST Tuberculin Skin Test
IGRA Interferon Gamma Release Assay
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