ABSTRACT

Objective: To evaluate tuberculin test results and relate them to the presence or absence of active tuberculosis, as well as to CD4+ and CD8+ T-lymphocyte counts. Method: The charts of 802 patients with acquired immunodeficiency syndrome treated between August of 1985 and March of 2003 were reviewed. Of the 185 patients submitted to tuberculin tests (23.1%), 107 (57.8%) were male, and 78 (42.2%) were female. Patients were divided into two study groups: tuberculin test reactors (n = 28); and tuberculin test non-reactors (n = 157). Among the reactors, the mean age was 30.60 years, with a standard deviation of 6.62 years, compared with 34.45 years, with a standard deviation of 10.32 years, among the non-reactors. Results: Most of the individuals tested presented only a mild response to the tuberculin test. We found that, at the time of the test, the percentage of individuals with active tuberculosis was greater in the reactor group than in the non-reactor group. During the test period, 10 reactor group patients and 11 non-reactor group patients presented some clinical form of active tuberculosis. In addition, CD4+ T-lymphocyte counts were lower than 200 cells/mm3 in 6 reactor group patients and in 8 non-reactor group patients. Conclusion: Indurations greater than 5 mm were unrelated to higher absolute CD4+ T-cell counts.

Keywords: Tuberculin test; Acquired immunodeficiency syndrome; Tuberculosis; CD4-positive T-lymphocytes; CD8-positive T-lymphocytes

RESUMO

Objetivo: Avaliar os resultados do teste tuberculínico e relacioná-los com a presença ou não de tuberculose em atividade e com a contagem de linfócitos T CD4+/CD8+. Métodos: Foram revisados 802 prontuários de pacientes com síndrome da imunodeficiência adquirida atendidos no período de agosto de 1985 a março de 2003. Cento e oitenta e cinco pacientes realizaram o teste tuberculínico (23,1%) e, destes, 107 eram do sexo masculino (57,8%). A média de idade no grupo de reatores ao teste tuberculínico foi de 30,6 anos, com desvio-padrão de 6,62 anos, e entre os não reatores de 34,45 anos com desvio-padrão de 10,32 anos. Foram constituídos dois grupos de estudo: reatores ao teste tuberculínico, com 28 pacientes, e não reatores ao teste tuberculínico, com 157 pacientes. Resultados: Grande parte dos indivíduos foi pouco responsiva ao teste tuberculínico. Constatou-se, no grupo de reatores, maior porcentagem de indivíduos com tuberculose ativa à época da realização do teste, quando se comparou com os não reatores. Dez pacientes entre os reatores e onze entre os não reatores apresentavam alguma forma clínica de tuberculose em atividade à época da realização do teste, sendo que seis do primeiro grupo e oito do segundo tinham contagem de linfócitos T CD4+ menor que 200 células/mm3. Conclusão: Indurações maiores do que 5 mm não se relacionaram com contagens absolutas mais altas de células T CD4+.

Palavras-chave: Teste tuberculínico; Síndrome de imunodeficiência adquirida; Tuberculose; Linfócitos T CD4-positivos;

INTRODUCTION

According to the World Health Organization, one-third of the world population, approximately 1.7 billion people, are carriers of Mycobacterium tuberculosis, the infectious agent of tuberculosis. Of those, 5% to 10% will develop the disease, which is responsible for three million deaths per year.(1)

In Brazil, it estimated that 35 to 45 million people are infected with M. tuberculosis, and there are approximately 100,000 new cases every year. In a study conducted in 1996,(2) it was shown that tuberculosis was the most prevalent opportunistic infection among patients with acquired immunodeficiency syndrome (AIDS) in Rio de Janeiro. Between 1988 and 1996 in Brazil, among all the individuals with AIDS, 7.8% were co-infected with M. tuberculosis, of which 5.5% occurred among patients 13 years of age or older and 2.3% among those under 13.(3)

Tuberculosis affects patients in the early stages of infection with the human immunodeficiency virus (HIV), even prior to the development of severe immunosuppression.(1) With the development of AIDS, there are uncommon and severe presentations of the disease, such as disseminated and lymph node forms of tuberculosis.(4-6)

The purified protein derivative (PPD) test, a delayed-type hypersensitivity skin test, is used as an auxiliary method in the diagnosis of tuberculosis, as well as to identify cases in which chemoprophylaxis with isoniazid is indicated.(7-8) This test, if interpreted in isolation, identifies the infection but not active tuberculosis. Among immunocompetent patients, the diameter of the palpable induration distinguishes nonreactors (up to 4 mm) from weak reactors (5 to 9 mm). Induration equal to or greater than 10 mm indicates that the individual is a strong reactor. However, among HIV-positive individuals (including those with AIDS), an induration equal to or greater than 5 mm is considered a strong reaction.(7-8)

Individuals co-infected with HIV/M. tuberculosis normally respond to the PPD skin test as nonreactors, which is different from what one finds among patients with tuberculosis alone. Some authors(9) who studied individuals with pulmonary tuberculosis without AIDS found that 75% were strong reactors to the PPD test. Others(5) found that the majority of 339 individuals co-infected with the pulmonary, extrapulmonary or associated forms of tuberculosis were nonreactors to the PPD, which was in accordance with various other studies.(10-14) Differing from these, some authors(15)found similar proportions of individuals who were HIV seropositive (80%) or seronegative (93%) among those presenting indurations equal to or greater than 10 mm and active tuberculosis. The weaker response to the PPD test among HIV-positive individuals is certainly due to the alterations in the cellular immune response caused by the virus, which, as some other authors(16) have shown, can even lead to anergy. Those authors, who carried out delayed-type hypersensitivity skin tests, including the PPD test, reported anergy among 10% of HIV-positive individuals and found that these individuals had CD4+/mm3 T-cell counts equal to or greater than 500 cells/mm3 and that two-thirds of those had counts lower than 200 cells/mm3. In another study,(17) strong PPD test reactivity was observed in 108 (29%) of the 374 HIV-positive patients analyzed. In yet another study,(18) no relationship was found between diameter of the induration and CD4+ T-cell count.

The objective of the present study was to evaluate the skin reaction among HIV-positive individuals (including those with AIDS), to the tuberculin test and to relate that response to the number of peripheral T-cells with CD4+ and CD8+ markers, as well as to the presence or absence of clinically active tuberculosis.

METHODS

In order to select the patients, a retrospective study was conducted to evaluate the medical charts of 802 HIV-positive individuals (including those with AIDS), all of whom were treated in the Tropical Diseases Outpatient Clinic of the Botucatu School of Medicine of the Paulista State University between August of 1985 and March of 2003. The inclusion criterion was having had a PPD skin test.

The 185 patients who had had a PPD skin test were divided into two groups according to the skin reaction to the PPD test: the strong reactor group, composed of 28 patients (induration = 5 mm); and the nonreactor group, composed of 157 patients (induration < 5 mm).

The data collection was carried out during the review of the medical charts. The following variables were considered: age; gender; stage of HIV infection; current tuberculosis status; results of the PPD test; CD4+ T-cells/mm3; and CD8+ T-cells/mm3. The PPD tests and the T-cell counts were performed during the same period.

The diagnosis of tuberculosis was established based on positive sputum smear microscopy or positive culture of any biological material, with histopathological examination compatible with or showing the presence of bacilli, as well as on epidemiological history or radiological exams suggestive of the active form of the disease and on a response to specific treatment.

The PPD skin test using PPD-RT23 in the dose equivalent to 2 tuberculin units, which is part of the routine treatment of HIV-positive patients, was carried out by various professionals, all of whom were trained by the São Paulo State Department of Health. The results were read with a millimeter ruler and always by the same professional who had applied the antigen. No patient had performed the PPD skin test within the preceding six months.

The two groups were compared using the methods described below. For binary variables (yes/no and gender), the chi-square test or Fisher's exact test was utilized. For quantitative variables, the t-test for two independent samples was used. The calculated statistics were considered significant when values of p < 0.05 were found, p being the probability of drawing an erroneous conclusion regarding the significance.(19)

The study design was approved by the Ethics in Research Committee of the Paulista State University Botucatu School of Medicine.

RESULTS

Of the 185 patients who had the PPD test, 28 (15.1%) were strong reactors and 157 (84.9%) were nonreactors. Among the strong reactors, the induration of the tuberculin test varied from 5 to 25 mm. There were 107 male patients (58.6%) and 78 female patients (41.4%). There was a greater proportion of males in the nonreactor group ( 2 = 3.902; p < 0.05) (Table 1).





With respect to age, the mean reported among the strong reactors was 30.6 ± 6.62 years, which was significantly lower than the mean age among the nonreactors, 34.45 ± 10.32 years. (t = 2.57; p < 0.05) (Table 2).





Concerning the stage of the HIV infection, 25% of the strong reactors were asymptomatic, and the remainder had AIDS. Among the nonreactors, 42.6% were asymptomatic, and 57.4% had AIDS ( 2 = 3.102; p > 0.05).
The proportion of individuals with active tuberculosis at the time of the PPD skin test was significantly greater among the strong reactors. ( 2 = 19.379; p < 0.01) (Table 3).





The mean and standard deviation of the CD4+ T-cell counts were equivalent in both groups (t = 1.16; p > 0.10). The same occurred in relation to the CD8+ T-cells (t = 0.17; p > 0.50). For both CD4+ and CD8+ T-cells, the lower limit of the count was always lower among the nonreactors than among the strong reactors (Table 4).





DISCUSSION

In the present study, the groups were not homogeneous with respect to gender and age bracket. In Brazil, males have been predominant in the AIDS population since the onset of the epidemic, although the ratio of males to females has been falling over the years.(20)

The predominance of the 20-39 age bracket is in accordance with the distribution of AIDS cases reported in Brazil, although an increase in the number of cases in the 40-49 age bracket has been observed.(20) The aging of the epidemic has also been observed in the USA, where, over the last decade, the number of cases among adults aged 50 or more has quintupled.(21) In Brazil, it has increased from 6.2% in the 1980s to 9.4% in 2003,(22) a fact that might be explained by the universal access to antiretroviral treatment, which has resulted in greater life expectancy among individuals with AIDS.

The progressive depletion of CD4+ T-cells due to HIV infection leads to the loss of important immunological functions, favoring the advent of opportunistic infections and neoplastic diseases.(23) However, the functional decline can be observed even before a significant reduction in the number of CD4+ T-cells is detected.(24-25)

Therefore, CD4+ T-cells and the components of the cellular population resident in the lung, including alveolar microphages and fibroblasts infected early with HIV, impair pulmonary function, which makes the lung quite vulnerable to opportunistic infections such as tuberculosis.(26) The CD8+ T-cells also play a role in immunity against the etiologic agent of tuberculosis. Data from experimental models indicate that animals with low CD8+ T-cell counts are much more susceptible to infection with M. tuberculosis, specifically in the lungs.(26) These cells appear to be the source of interferon-g, a cytokine essential in the defense against M. tuberculosis, and therefore might be involved in the activation of macrophages.(26)

In the present study, there was no significant difference between the two groups in terms of mean CD4+ and CD8+ T-cell counts. In both groups, the mean CD4+ T-cell count was above 200 cells/mm3. Among the nonreactors, half of the patients had CD4+ T-cell counts > 200 cells/mm3.
In Africa, some authors(27) have found CD4+ T-cell counts among patients with AIDS/tuberculosis comorbidity that are quite similar to those found in the present study. These authors suggested that the high frequency of tuberculosis among individuals with AIDS and those with mild immunodeficiency was related to the high prevalence of M. tuberculosis infection in the African population. The same occurred in Brazil, where the incidence and prevalence of the infection are rather significant.(22) In addition, due to its relatively high virulence, M. tuberculosis manifests itself during the course of the immunosuppression, earlier than do other opportunistic agents.(15) A different situation was found in the USA, where the epidemiology is considerably different from that of Brazil and Africa. Among individuals with AIDS/tuberculosis in the USA, much lower CD4+ T-cell counts were reported.(6)

The relationship between the number of CD4+ T-cells/mm3 and the skin reaction to the PPD test remains an unresolved matter. In the present study, 84.9% of the analyzed patients presented anergy to the tuberculin test, despite 16.6% of them having CD4+ T-cell counts = 500/mm3 and 50.3% of them having counts above 200/mm3. Even among reactors to PPD test, among which only 10.7% had CD4+ T-cell counts = 500/mm3, the diameter of induration varied from 5 mm to 25 mm. These data are similar to those of another study,(18) in which no relationship was observed between the size of the PPD test induration and CD4+ T-cell counts. Despite the mean counts not having differed, it should be emphasized that the minimum CD4+ and CD8+T-cell counts were lower among nonreactors than among strong reactors. Another study,(17) which evaluated 374 HIV-positive individuals, found 108 strong reactors with CD4+ T-cell counts equivalent to those of nonreactors. In contrast, other authors(16) reported anergy in only 10% of HIV-positive individuals with CD4+ T-cell counts = 500 cells/mm3, compared with approximately 60% in those with CD4+ T-cell counts = 200 cells/mm3.

In the present study, there might have been a significantly greater proportion of patients with active tuberculosis among HIV-positive strong reactors to the PPD test. However, there is no universal agreement on the value of skin reaction to the PPD test as an indicator of active tuberculosis. Some authors(29) have suggested that response not be used as a criterion for identifying cases of HIV seropositivity in which antituberculosis treatment is indicated. Others(17) have reported active tuberculosis among 5.4% of the individuals presenting HIV seropositivity no reaction to the PPD test. Still others(15) have reported induration = 10 mm among 80% of HIV-positive tuberculosis patients, compared with 30% of those with tuberculosis and without HIV. According to some authors,(1) the sensitivity to the tuberculin test among individuals presenting HIV seropositivity is inversely proportional to the degree of immunosuppression. In one study,(30) indurations = 10 mm were reported among 40% to 60% of active tuberculosis patients presenting HIV seropositivity and having been previously asymptomatic, compared with 10% to 30% of AIDS patients.

In conclusion, the results of the present study are in agreement with those of other studies showing that most HIV-positive individuals do not respond to the PPD skin test, and that the intensity of the response is unrelated to CD4+ T-cell counts. Although there is no consensus on the role of PPD skin tests in the diagnosis of active tuberculosis among AIDS patients, the results of the present study show that strong reactors to the test were more often diagnosed with tuberculosis.

REFERENCES

1. Shafer RW, Edlin BR. Tuberculosis in patients infected with human immunodeficiency virus: perspective on the past decade. Clin Infect Dis. 1996;22(4):683-704.
2. Silva G, Barreira D. Tuberculosis as opportunistic disease among AIDS in the city of Rio de Janeiro [abstract MO.C 1643]. In: 11° International Conference on Aids. Meeting. Rio de Janeiro, 7-12 Jul. 1996.
3. Brasil. Ministério da Saúde. Programa Nacional de DST/ AIDS. Aids Bol Epidemiol. 1999;12(4):43-4.
4. Sunderam G, McDonald RJ, Maniatis T, Oleske J, Kapila R, Reichman LB. Tuberculosis as a manifestation of the Acquired Immunodeficiency Syndrome (AIDS). JAMA. 1986;256(3):362-6.
5. Bethlem N, Souza GRM, Bethlem EP, Silva WAE. SIDA/AIDS e tuberculose no Brasil. Arq Bras Med. 1990;64(1):28-32.
6. Whalen C, Horsburgh Jr CR, Hom D, Lahart C, Simberkoff M, Ellner J. Site of disease and opportunistic infection predict survival in HIV-associated tuberculosis. AIDS. 1997;11(4):455-60.
7. Brasil. Ministério da Saúde. Centro Nacional de Epidemiologia. Coordenação de Pneumologia Sanitária. Manual de normas para o controle da tuberculose. Brasilia: Ministerio da Saude; 1995. 43p.
8. Dalcolmo MP, Macedo EA, Menezes LL, Paiva MAS, Sant'Anna CC. Prevenção da tuberculose: vacinação BCG e quimioprofilaxia. J Pneumol. 1993;19(1):60-2.
9. Nash DR, Douglass JE. Anergy in active pulmonary tuberculosis. A comparison between positive and negative reactors and an evaluation of 5 TU and 250 TU skin test doses. Chest. 1980;77(1):32-7.
10. Canessa PA, Fasano L, Lavecchia MA, Torraca A, Schiattone ML. Tuberculin skin test in asymptomatic HIV seropositive carriers. Chest. 1989;96(5):1215-6.
11. Colebunders RL, Lebughe I, Nzila N, Kalunga D, Francis H, Ryder R, et al. Cutaneous delayed-type hypersensitivity in patients with human immunodeficiency virus infection in Zaire. J Acquir Immune Defic Syndr. 1989;2(6):576-8.
12. Graham NM, Nelson KE, Solomon L, Bonds M, Rizzo RT, Scavotto J, et al. Prevalence of tuberculin positivity and skin test anergy in HIV-1-seropositive and -seronegative intravenous drug users. JAMA. 1992;267 (3):369-73. Comment in: JAMA. 1992;267(3):409-10. JAMA. 1992;267(18):2471-2. JAMA. 1992;267(18):2472.
13. Markowitz N, Hansen NI, Wilcosky TC, Hopewell PC, Glassroth J, Kvale PA, et al. Tuberculin and anergy testing in HIV-seropositive and HIV-seronegative persons. Pulmonary Complications of HIV Infection Study Group. Ann Intern Med. 1993;119(3):185-93. Comment in: Ann Intern Med. 1993;119(3):241-3.
14. Toledo Jr A, Machado LG, Carvalhais LM, Tupinambás U, Bello AP, Pádua CJ, et al. PPD tuberculin test and skin test anergy in an HIV infection high risk population, in Belo Horizonte, Brazil [abstract B.1089]. In: 11° International Conference on Aids. Meeting. Rio de Janeiro, 7-12 July 1996.
15. Theuer CP, Hopewell PC, Elias D, Schecter GF, Rutherford GW, Chaisson RE. Human immunodeficiency virus infection in tuberculosis patients. J Infect Dis. 1990;162(1):8-12.
16. Maas JJ, Foudraine NA, Schellekens PT, Mensen ME, Veenstra J, Roos MT, et al. Reliability of tuberculin purified derivative skin testing and delayed-type hypersensitivity skin test anergy in HIV-infected homosexual men, at risk of tuberculosis.AIDS. 1999;13(13):1784-5.
17. Moreno S, Baraia-Etxaburu J, Bouza E, Parras F, Perez-Tascon M, Miralles P, et al. Risk for developing tuberculosis among anergic patients infected with HIV. Ann Intern Med. 1993;119(3):194-8. Comment in: Ann Intern Med. 1993;119(3):241-3.
18. Moreno S, Baraia-Etxaburu J, Miralles P, Berenguer J, Cosin J, Bernaldo de Qurios JC. Tuberculin reaction size in patients infected with HIV. AIDS. 1995;9(11):1289-90.
19. Curi PR. Metodologia e análise da pesquisa em ciencias biológicas. 2a ed. Botucatu: Tiponic; 1998. 215 p.
20. Brasil. Ministério da Saúde. Programa Nacional de DST/AIDS. Aids Bol Epidemiol. 2001/2002;15(1):14-5.
21. Levy JA, Ory MG, Crystal S. HIV/AIDS interventions for midlife and older adults: current status and challenges. J Acquir Immune Defic Syndr. 2003;33(Suppl.2):S59-67.
22. Brasil. Ministério da Saúde. Programa Nacional de DST/AIDS. Perspectiva histórica das definições de caso de Aids no Brasil vigente até 2003. Aids Bol Epidemiol [periódico na Internet]. 2003 [citado 2006 Ago 21];17(1):27-8. Disponível em: http://www.aids.gov.br/final/biblioteca/boletim_dezembro_2003/bol_dezembro_2003.pdf
23. Clerici M, Shearer GM. A TH1-->TH2 switch is a critical step in the etiology of HIV infection. Immunol Today. 1993;14(3):107-11. Comment in: Immunol Today. 1993;14(4):190-1. Immunol Today. 1993;14(8):417-8. Immunol Today. 1993;14(11):568-9. Immunol Today. 1994;15(2):90-1. Immunol Today. 1995;16(10):501.
24. Clerici M, Stocks NI, Zajac RA, Boswell RN, Bernstein DC, Mann DL, et al. Interleukin-2 production used to detect antigenic peptide recognition by T-helper lymphocytes from asymptomatic HIV-seropositive individuals. Nature. 1989;339(6223):383-5.
25. Miedema F, Petit AJ, Terpstra FG, Schattenkerk JK, de Wolf F, Al BJ, et al. Immunological abnormalities in human immunodeficiency virus (HIV), infected asymptomatic homosexual men. HIV affects the immune system before CD4+ T helper cell depletion occurs. J Clin Invest. 1988;82(6):1908-14.
26. Orme IM, Cooper AM. Cythokine/chemokine cascades in immunity to tuberculosis. Immunol Today. 1999;20(7):307-12.
27. Ackah AN, Coulibaly D, Digbeu H, Diallo K, Vetter KM, Coulibaly IM, et al. Response to treatment, mortality, and CD4 lymphocyte counts in HIV-infected persons with tuberculosis in Abidjan, Cote d'Ivoire. Lancet. 1995;345(8950):607-10.
28. Ministério da Saúde. Fundação Nacional de Saúde. Controle da tuberculose: uma proposta de integração ensino-serviço. Rio de Janeiro: FUNASA; 2002 [citado 2006 Ago 21]. Disponível em: http://dtr2001.saude.gov.br/bvs/publicacoes/ ensino_servico.pdf
29. Chin DP, Osmond D, Page-Shafer K, Glassroth J, Rosen MJ, Reichman LB, et al. Reability of anergy skin testing in persons with HIV infection. The pulmonary Complications of HIV Infection Study Group. Am J Respir Crit Care Med. 1996;153(6 Pt 1):1982-4. Comment in: Am J Respir Crit Care Med. 1997;155(4):1490.
30. Barnes PF, Bloch AB, Davidson PT, Snider DE Jr. Tuberculosis in patients with human immunodeficiency virus infection. N Engl J Med. 1991;324(23):1644-50. Comment in: N Engl J Med. 1991;325(26):1882-4.
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* Study carried out at the Botucatu School of Medicine of the "Julio Mesquita Filho" Universidade Estadual Paulista (UNESP, Paulista State University), Botucatu, Brazil.
1. Assistant Professor and Ph.D from the Botucatu School of Medicine of the "Julio Mesquita Filho" Universidade Estadual Paulista (UNESP, Paulista State University), Botucatu, Brazil
2. Adjunct Professor at theUniversidade Federal do Ceará (UFC, Federal University of Ceara), Fortaleza, Brazil
3. Adjunct Professor of the Department of Tropical Diseases and Diagnostic Imaging at the Botucatu School of Medicine of the Universidade Estadual Paulista (UNESP, Paulista State University), Botucatu, Brazil
4. Professor Emeritus at the Botucatu School of Medicine of the "Julio Mesquita Filho" Universidade Estadual Paulista (UNESP, Paulista State University), Botucatu, Brazil
5. Undergraduate student at the Botucatu School of Medicine of the "Julio Mesquita Filho" Universidade Estadual Paulista (UNESP, Paulista State University), Botucatu, Brazil
Correspondence to: Lenice do Rosário de Souza. Departamento de Doenças Tropicais. Faculdade de Medicina de Botucatu - UNESP. Distrito de Rubião Júnior, s/n - CEP: 18618-970, Botucatu, SP, Brasil. Email: lsouza@fmb.unesp.br
Submitted: 16 May 2005. Accepted, after review: 16 December 2005.