Around 2 billion people suffer from latent mycobacterium tuberculosis infection (LTBI) (1). Prevalence of LTBI in health care workers (HCW) in middle and low-income countries ranges between 33 and 79% (2) and in high-income countries it is not higher than 55% (3). Due to professional exposure to tuberculosis patients, health care workers are at increased risk of tuberculosis infection.
LTBI diagnosis has been made for over last hundred years by in vivo method, tuberculin skin test (TST). TST is based on measuring reaction of postponed oversensitivity after a mixture of non-specified mycobacterium antigens (PPD, purified protein derivative) has been injected into the inner surface of the forearm. PPD is used to determine total effect of professional and non-professional exposure to tuberculosis bacillus and the result of reaction is influenced by factors like BCG vaccination (Calmette-Guerin Bacillus) and previous exposure to non-tuberculosis mycobacteria (4,5). Heterogeneity of PPD-solution, subjectivity at reaction reading and different cut-off values for determination of positive TST diminish its specificity. No positive control samples were used in the test and therefore false negative results were possible as consequence of immunosuppressive conditions (6). In countries like Croatia, where BCS vaccination is conducted regularly, TST can give false results.
A new approach to LTBI detection is based on whole human blood ex vivo assays which determine IFN-γ released from T-lymphocytes after their prolonged exposure to M. tuberculosis specific antigens. These antigens distinguish M. tuberculosis from the majority of other mycobacteria (7). This fact specifically contributes to higher specificity of this assay compared to TST. By using IFN-γ releasing assay (IGRA, interferon-gamma release assay) BCG-vaccinated individuals can be excluded, which is not the case with TST. Patients suffering from diabetes mellitus type 1 often have impaired cell immunity and are prone to contagious diseases (8). The aim of this study is to show a LTBI case in a 46 year-old female health care worker (a nurse at the Department for Tuberculosis in young children) suffering from diabetes mellitus type 1. Up to our knowledge, this is the first report of IGRA application in revealing LTBI in an individual who is professionally exposed to mycobacterium tuberculosis and at the same time suffers from type 1 diabetes mellitus.
A 46 year old female health care worker (a nurse) has been working for 26 years at the Department for Tuberculosis in young children. During the last 20 years she has been suffering from diabetes mellitus type 1. Because of the close contact with tuberculosis patients, she underwent a routine screening for tuberculosis infection.
The subject was BCG vaccinated (Bacille Calmette-Guerin) and has a BCG scar. At admission she was not acutely ill, she had normal body temperature, was eupneic and lung auscultation showed normal breathing. TST was negative (induration diameter 6 mm; reading after 72 hours; 2 tuberculin RT-23 units, Statens Serum Institute, Copenhagen, Denmark). Ex vivo IFN-γ test result was positive (≥ 0.35 kIU/L) and positive mitogen control was normal (≥ 0.50 kIU/L). The subject had elevated concentrations of glucose, IgA and hsCRP and elevated erythrocyte sedimentation rate (Table 1).
Table 1. Results of laboratory analyses
Venous blood is sampled before TST. Determination procedure is carried out in two phases:
Activation of patient’s whole blood lymphocytes with specific mycobacterium tuberculosis antigens (test tube contains three antigens - ESAT-6, CFP10 and TB7.7). At the same time blood is sampled for positive (test tube with phytohemagglutinin) and negative control (test tube with heparin). All three samples are incubated over night (22 hours) at 37°C, and plasma is thereafter separated and stored at 4°C until analysis (for max 7 days).
IFN-γ concentration determination by ELISA assay with QuantiFERON-TB Gold In Tube (Cellestis Ltd., Victoria, Australia) commercial assay. Test result is displayed as positive if IFN-γ concentration is higher than the cut-off value of ≥ 0.35 kIU/L.
Only IFN-γ releasing assay (IGRA) indicated the LTBI presence in the nurse who has been for years exposed to tuberculosis patients, while TST result was false negative. Despite diabetes mellitus over many years, active tuberculosis has not developed. From other test results, attention should be drawn to elevated glucose concentration as an indicator of poor diabetes control, and to elevated concentration of inflammatory markers. Elevated IgA concentration can be found in serum in approximately 23% of diabetes mellitus patients, especially if disease has not been treated longer than 10 years (9), which is the case with our female health care worker, or if diabetic nephropathy develops (10). Elevated hsCRP concentration points to latent inflammation which is characteristic for diabetes mellitus patients (11) or to a cardiovascular risk (12).
This case report opens two discussion topics: in vivo and ex vivo test result values for detecting tuberculosis infection in individuals professionally exposed to infectious agent, and relation between tuberculosis infection and diabetes mellitus.
British guidelines for tuberculosis diagnosis and treatment suggest initial screening by TST and positive result confirmation by some of IGRA assays (13). According to US guidelines, the newer IGRA version (QuantiFERON-TB Gold) can completely replace TST assay and it is not necessary to perform both assays simultaneously (14). However, one should bear in mind the diagnostic value of these assays. TST diagnostic value depends on the cut-off value determination that distinguishes positive from negative results (15), and on previous exposure to non-tuberculosis mycobacteria (4). For both assays, population vaccination is very important (16-18). BCG vaccination influences TST test results especially in individuals < 40 years old in countries with low tuberculosis incidence (19). BCG-vaccinated individuals with TST > 11 mm can be considered infected (20). A positive TST result after infection with M.tuberculosis often remains permanently positive (“oncepositive, no longer useful”) (21,22).IFN-γ concentration determination is a better indicator in vaccinated individuals (18) and in individuals longer exposed to M. tuberculosis (23) than TST because it points to acute exposure to specific tuberculosis antigens. Result of the IGRA in people with LTBI showed sensitivity of 90% and specificity of 98% (24). Specificity of IGRA is enhanced by introducing more mycobacterial antigens in the test tube. Test results are simple to interpret when they are comparable. In most studies, concordance between TST and IGRA results could vary between 60-90% (25). In the cases where TST is positive but IGRA is negative, discrepancy could be assigned to prior BCG vaccination (25). Reverse discrepancy (TST negative; IGRA positive) has also been documented in health care workers (26). Agreement of the new generation of IFN tests (Quantiferon-TB Gold, used in the present study) with TST is 79-94 % (27). However, in individuals with impaired cell immunity (patients suffering from malignant diseases, diabetes mellitus, chronic kidney disorder, HIV or on immunosuppressive therapy), results of these assays do not have to be comparable (28). Kobashi et al. showed that immunocompromised patients had negative skin test and positive IGRA test result (23), which is also the case in our study. Therefore, in individuals with impaired immunity, in vivo and ex vivo assays do not have the same diagnostic value as in healthy individuals.
This case report confirms the foregoing statement. It showed false negative skin test result and positive IGRA test result. Such outcome confirmed that, in spite of type 1 diabetes over many years, the IFN-γ releasing reaction toward specific tuberculosis antigens is still preserved. At the same time, by examining cell immunity by IFN-γ determination after phytohemagglutinin-mediated lymphocyte activation, we avoided a possible false negative result. Connection between tuberculosis and diabetes mellitus is a fact known for over 2000 years (29). Already in the 5th century, tuberculosis was described as a complication of diabetes mellitus. Diabetic patients are prone to infections (8) and therefore also to mycobacterium tuberculosis infection. Although there is not sufficient knowledge about the way diabetes impairs the immune system, it seems that this weak immune ability can be attributed to hyperglycemia (30). A study conducted on diabetic mice infected with m. tuberculosis (31) and investigation of the IFN-γ role in individuals suffering from diabetes mellitus also pointed to this causal connection (32). Diabetic mice had inflammatory changes in lungs and at the same time lowered IFN-γ production (31), and individuals with diabetes mellitus were prone to tuberculosis because of IFN-γ deficiency needed for initial growth inhibition of m. tuberculosis (32). Hence we can conclude that, in order to detect low IFN-γ production and consequent susceptibility to active tuberculosis, ex vivo IFN-γ assay should be included in the annual check-up of our subject.
Potential conflict of interest
1. World Health Organization: GlobalTuberculosis Control: Surveilleance, Planning, Financing. WHO report 2005. Geneva, Switzerland, 2005.
2. Joshi R, Reingold AL, Menzies D, Pai M. Tuberculosis among health-care workers in low- and middle-income countries: a systematic review. PLOS Medicine 2006;3:2376-91.
3. Seidler A, Nienhaus A, Diel R. Review of epidemiological studies on the occupational risk of tuberculosis in low-incidence areas. Respiration 2005;2:431-6.
4. Snider DE. Bacille Calmette-Guerin vaccinations and tuberculin skin test. J Am Med Assoc 1985;253:3438-9.
5. Daley AJ, Isaacs D. Differential avian and human tuberculin skin testing in non-tuberculous mycobacterial infection. Arch Dis Child 1999;80:377-9.
6. Huebner RE, Schein MF, Bass JBJ. The tuberculin skin test. Clin Infect Dis 1993;17:968-75.
7. Mori T, Sakatani M, Yamagishi F, Takashima T, Kawabe Y, Nagao K, et al. Specific detection of tuberculosis infection. An interferon-γ-based assay using new antigens. Am J Respir Crit Care Med 2004;170:59-64.
8. Joshi N, Caputo GM. Weitekamp MR, Karchmer AW. Infections in patients with diabetes mellitus. N Engl J Med 1999;341:1906-12.
9. Rodriguez-Segade S, Camina MF, Carnero A, Lorenzo MJ, Alban A, Quinteiro C, et al. High serum IgA concentrations in patients with diabetes mellitus: agewise distribution and relation to chronic complications. Clin Chem 1996;42:1064-7.
10. Kanauchi M. Serum IgA levels in patients with diabetic nephropathy and IgA nephropathy superimposed on diabetes mellitus. Diabetes Research and Clinical Practice 2000;48:113-8.
11. King DE, Mainous AG, Buchanan TA, Pearson WS: C-reactive protein and glycemic control in adults with diabetes. Diabetes Care 2003;26:1535–9.
12. Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM. C-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus. JAMA 2001;286:327-34.
13. NICE - National Institute for Health and Clinical Excellence. Clinical Guideline 33, Tuberculosis: clinical diagnosis and management of tuberculosis and measurement for its prevention and control. NICE, London, UK, 2006. Available at http://www.nice.org.uk.
14. Mazurek GH, Hereb J, Lobue P, Iademarco MF, Metchock B, Vernon A. Guideline for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR Recomm Rep 2005;54(RR-15):49-55.
15. Ozdemir D, Annakkaya AN, Tarhan G, Sencan I, Cesur S, Balbay O, et al. Comparison of the tuberculin skin test and the Quantiferon test for latent Mycobacterium tuberculosis infections in health care workrs in Turkey. Jpn J Infect Dis 2007;60:102-5.
16. Nienhaus A, Schablon A, Le Bâcle C, Siano B, Diel R. Evaluation of the interferon-γ release assay in healthcare workers. Int Arch Occup Environ Health 2008;81:295-300.
17. Harada N, Nakajima Y, Higuchi K, Sekiya Y, Rothel J, Mori T. Screening for tuberculosis infection using whole-blood interferon- γ and Mantoux testing among Japanese healthcare workers. Infect Control Hosp Epidemiol 2006;27:442-8.
18. Kang YA, Lee HW, Yoon HI, Cho BL, Han SK, Shim YS, et al. Discrepancy between the tuberculin skin test and the whole-blood interferon-γ assay for the diagnosis of latent tuberculosis infection in an intermediate tuberculosis-burden country. JAMA 2005;293:2756-61.
19. Tissot F, Zanetti G, Francioli P, Zellweger JP, Zysset F. Influence of bacille Calmette-Guérin vaccination on size of tuberculin skin test reaction: to what size? Clin Infect Dis 2005;40:211-7.
20. Bugiani M, Borraccino A, Migliore E, Carosso A, Piccioni P, Cavallero M, et al. Tuberculin reactivity in adult BCG-vaccinated subjects: a cross-sectional study. Int J Tuberc Lung Dis 2003;7:320-6.
21. Arend SM, Thijsen SFT, Leyten EMS, Bouwman JJM, Franken WPJ, Koster BFPJ, et al. Comparison of two interferon-γ assays and tuberculin skin test for tracing tuberculosis contacts. Am J Resp Crit Care Med 2007;175:618-27.
22. Menzies D. Interpretation of repeated tuberculin tests: boosting, conversion, and reversion. Am J Respir Crit Care Med 1999;159:15–21.
23. Kobashi Y, Obase Y, Fukuda M, Yoshida K, Miyashita N, Fujii M, et al. Usefulness of QuantiFERON TB-2G, a diagnostic method for latent tuberculosis infection, in a contact investigation of health care workers. Intern Med 2007;46;1543-9.
24. Streeton JA, Desem N, Jones SL. Sensitivity and specificity of gamma interferon blood test for tuberculosis infection. Int J Tuberc Lung Dis 1998;2:435-50.
25. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: Part I. Latent tuberculosis. Expert Rev Mol Diagn 2006;6:413-22.
26. Pai M, Gokhale K, Joshi R, Dogra S, Kalantri S, Mendiratta DK. Mycobacterium tuberculosis Infection in health care workers in rural India. Comparison of a whole-blood - γ assay with tuberculin skin testing. JAMA 2005;293:2746-55.
27. Madariaga MG, Jalali Z, Swindells S. Clinical utility of interfereon gamma assay in the diagnosis of tuberculosis. J Am Board Fam Med 2007;20:540-7.
28. Kobashi Y, Mouri K, Obase Y, Fukuda M, Miyashita N, Oka M. Clinical evaluation of QuantiFERON TB-2G test for immunocompromised patients. Eur Respir J 2007;30:945-50.
29. Broxmeyer L. Diabetes mellitus, tuberculosis and the mycobacteria: Two millenia of enigma. Med Hypotheses 2005;65:433-9.
30. Gupta S, Koirala J, Khardori R, Khardori N. Infections in diabetes mellitus and hyperglycemia. Inf Dis Clin North Am 2007;21:617-38.
31. Martens FW, Arikan MC, Lee J, Ren F, Greiner D, Kornfeld H. Tuberculosis susceptibiliy of diabetic mice. Am J Respir Cell Mol Biol 2007;37:518-24.
32. Stalenhoef JE, Alisjahbana B, Nelwan EJ, van der Ven-Jongerkrijg J, Ottenhoff TH, van der Meer JW, et al. The role of interferon-gamma in the increased tuberculosis risk in type 2 diabetes mellitus. Eur J Clin Microbiol Infect Dis 2008;27:97-103.