Leprosy and confinement due to leprosy show high association with hepatitis C in Southern Brazil

Acta Tropica 97 (2006) 88–93

Leprosy and confinement due to leprosy show high association with hepatitis C in Southern Brazil Anna Carolina de Moraes Braga a,∗ , Iara J. Messias Reason b,c , Eliane Ces´ario Pereira Maluf d , Elizabete Regina Vieira a a

d

Servi¸co de Hemoterapia, Hospital de Cl´ınicas da Universidade Federal do Paran´a, Curitiba, Paran´a, Brazil b Laborat´ orio de Imunopatologia, Departamento de Patologia M´edica, Hospital de Cl´ınicas da Universidade Federal do Paran´a, Curitiba, Paran´a, Brazil c Institut of Tropical Medicine, University of T¨ ubingen, Germany Departamento de Cl´ınica M´edica, Hospital de Cl´ınicas da Universidade Federal do Paran´a, Curitiba, Paran´a, Brazil Received 13 April 2005; received in revised form 8 September 2005; accepted 9 September 2005 Available online 2 November 2005

Abstract Leprosy is a disease, which is accompanied by cellular immunity defects, which may increase the susceptibility of patients in developing co-infections. The association of leprosy with hepatitis C virus (HCV) infection, human immunodeficiency virus types 1 and 2 (HIV 1 + 2) infection and human T-lymphotropic virus types I and II (HTLV I + II) infection have previously been described in different populations. In this study, the prevalence of these infections was determined in 199 Southern Brazilian leprosy patients and in 681 matched controls. Antibodies to HCV were positive in 3.52% of the patients (7/199) and in 0.15% of the controls (1/681; odds ratio (OR) = 24.79; 95% CI = 3.03–202.74; p = 0.0002). An increased risk of HCV infection was observed in institutionalized patients (OR = 14.95; 95% CI = 1.76–127.03; p = 0.004) and in the lepromatous form of the disease (OR = 7.67; 95% CI = 0.43–136.62; p = ns). Anti-HIV 1 + 2 antibodies were positive in only one out-patient (1/199; 0.50%) and in none of the controls (0/681; OR = 3.43; 95% CI = 0.21–55.16; p > 0.05). No leprosy patient was positive for anti-HTLV I + II antibodies. These results demonstrate an increased prevalence of HCV infection in leprosy patients from South Brazil and that both institutionalization and lepromatous form of the disease confer higher risk to HCV infection. These data emphasizes the importance of monitoring hepatitis C and leprosy interactions and the need of special care to institutionalized and lepromatous patients in preventing HCV co-infection. © 2005 Elsevier B.V. All rights reserved. Keywords: Leprosy; Hepatitis C; Human immunodeficiency virus; Human T lymphotropic virus; Brazil; Co-infection

1. Introduction Brazil is second only to India as the country with the highest endemicity of leprosy in the world, with a prevalence of 4.1 cases/10,000 inhabitants in 2003 (World

∗

Corresponding author. Tel.: +55 41 3360 7887; fax: +55 41 3360 7887. E-mail address: [email protected] (A.C. de Moraes Braga). 0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.09.003

Health Organization, 2004). The wide spectrum of clinical manifestations in leprosy appears to be determined by a complex and distinct immunologic interaction between the host and Mycobacterium leprae. The lepromatous form, characterized by the low resistance pole of Hansen disease, is accompanied by cellular immune defects that may increase the susceptibility of these patients in developing co-infections. The other pole of the disease represented by the tuberculoid form, presents a vigorous cellular response to M. leprae, which

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controls bacillus dissemination protecting the individual from the disease (Goulart et al., 2002). Although there are several studies showing an increased prevalence of different infections in leprosy, the results are divergent, which probably is due to the different parameters used such as: local epidemiology, sex and age distribution, socio-economic condition of patients and controls and methodology (Andrade et al., 1997). The association between leprosy and hepatitis C virus (HCV), leprosy and human immunodeficiency virus types 1 and 2 (HIV 1 + 2) and leprosy and human Tlymphotropic virus types I and II (HTLV I + II) infection have been described in different countries around the world. In Senegal, Cisse et al. (1990) demonstrated a prevalence of 85.4% of anti-HCV in leprosy patients, determined by an enzyme immunoassay (EIA). Later, the same authors using confirmatory tests such as immunoblot observed positivity in only 7.3% of the same samples. Denis et al. (1991) demonstrated 4.0–6.0% of anti-HCV positivity in Senegal, Congo and other African countries, presenting a significant difference when compared to the control group. High prevalence of HCV infection in leprosy patients was also reported in Ethiopia and in Yemen (Frommel et al., 1993). In Japan, a highly significant prevalence of anti-HCV was observed in leprosy patients from a leprosarium, using EIA (30.0%) and RNA test (18.0%), when compared to controls (1.2% EIA and 1.0% RNA test) (Egawa et al., 1996). According to the World Health Organization (2001), African countries present the highest prevalence rates of HIV infection in the world ranging from 15.0 to 39.0%. The positivity of anti-HIV in leprosy patients was reported to be 3.2% in Ethiopia (Tekle-Haimanot et al., 1991), 1.15% in Senegal (Blum et al., 1992), 6.0% in Nigeria (Awofeso, 1995) and 8.0% in Nairobi, Kenya (Munyao et al., 1994). On the other hand, HTLV-1 infection was shown to occur endemically in some countries such as Japan and Ethiopia with an association with leprosy, varying from 0.4% in Ethiopia (Tekle-Haimanot et al., 1991) to 25.8% in Japan (Hanada et al., 1989). In Brazil, there is one study describing the association of leprosy and HIV, in Rio de Janeiro, with a seroprevalence of 0.29% (Andrade et al., 1997). There is also one study on the prevalence of leprosy and hepatitis C in the central region, which reported a positivity of anti-HCV of 1.8% (Rosa et al., 1996). On the other hand, there are no data on the association of HTLV infection and leprosy in Brazil.

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In this study, the prevalence of co-infection by HCV, HIV 1 + 2 and HTLV I + II was determined in leprosy patients and controls from Southern Brazil. 2. Materials and methods 2.1. Patients and controls One hundred and ninety-nine leprosy patients (mean age of 52 ± 16 years, range 18–94 years; 122 male and 77 female) from Curitiba, Southern Brazil were studied. Patients were classified according to the criteria of Ridley and Jopling (1966) and were included in the study according to WHO definition, in which patients presents one or more of the following features: hypopigmented or reddish skin lesion(s) with definite loss of sensation; involvement of the peripheral nerves, as demonstrated by definite thickening with loss of sensation; and skin smear positive for acid-fast bacilli. One hundred and nineteen patients were diagnosed as presenting the lepromatous form of the disease, 15 the tuberculoid and 65 the other forms (indeterminate and borderline). Among the patients studied, 138 were out-patients (Hospital de Cl´ınicas of Federal University and Secretaria de Sa´ude P´ublica of Paran´a) and 61 were in-patients (Hospital de Dermatologia Sanit´aria of Paran´a). Patients with advanced clinical manifestations were typically in the institution for many years since being abandoned by their families, whereas others were admitted to the institution for treatment of different clinical conditions. The out-patients have not been in confinement, since they underwent anti-leprosy drug treatment early and the disease was under control. Some out-patients were admitted to other hospitals during short periods of time. As controls, 681 blood donors from the same geographic region (mean age of 45 ± 12 years, range 18–65 years; 412 male and 160 female) were consecutively selected by their donation number (Table 1). Both patients and controls were selected in 2002 and 2003. This study was approved by the ethics committee of the Clinic Hospital and Health State Department, and all subjects included were asked to fill in an informed consent for their participation in the investigation. A standardized questionnaire was used to determine the history of the patients regarding possible transfusion and other infectious diseases. 2.2. Serum samples Ten millilitre of venous blood was collected from each subject and separated by centrifugation. Serum was kept at −20 ◦ C until tested for anti-HCV (a-HCV), anti-HIV

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Table 1 Demographical, clinical characteristics and prevalence of HCV, HIV1 + 2 and HTLV I + II infection of leprosy patients and healthy controls from Southern Brazil Patients

Assistency form (out-patients/in-patients)

Disease form (lepromatous/others)

Controls (M/F)

Age (years)

Sex (M/F)

15–24 25–49 ≥50

4/5 42/33 76/39

9/0 68/7 61/54

4/5 41/34 74/41

16/20 168/132 228/117

Total

122/77

138/61

119/80

412/269

1 + 2 (a-HIV 1 + 2) and anti-HTLV I + II (a-HTLV I + II) antibodies.

parison of odds ratios with Haldane correction with the respective 95% confidence limits.

2.3. Evaluation of anti-HCV, anti-HIV 1 + 2 and anti-HTLV I + II antibodies

3. Results 3.1. Anti-HCV antibodies

Anti-HCV antibodies were detected by a thirdgeneration immunoassay with antigens of core (structural proteins) and NS3, NS4 and NS5 (nonstructural proteins) (Murex anti-HCV, Version 4.0, Murex Biotech S.A. Ltd., Kyalami, South Africa). Positive samples were retested for confirmation with the same commercial kit. All confirmed a-HCV-positive samples were retested by an enzyme immunoblot assay which utilizes recombinant HCV-encoded antigens (c33c and NS5) and synthetic HCV-encoded peptides (5-1-1p, c100p and c22p) immobilized as individual bands onto test strips (CHIRON RIBA HCV 3.0 SIA, Chiron Corporation, Emerville, USA) and the samples were considered positive when both results showed positivity. IgG and IgM antibodies to HIV 1 + 2 were detected by an enzyme immunoassay (Murex HIV-1.2.O, Murex Biotech Ltd., Temple Hill, UK). Repeated-positive samples were retested through a microparticle enzyme immunoassay (MEIA) for HIV-1 (groups M and O) and HIV-2 (AxSYM® HIV (1/2) gO, Abbott GmbH, Wiesbaden, Germany) and by indirect immunofluorescent (IFI) test and they were considered HIV positive when reactive in all tests. HTLV I + II antibodies were determined using an EIA based on recombinant proteins of HTLV I and HTLV II transmembrane proteins and synthetic peptides of external membrane proteins of both virus (Murex HTLV I + II, Murex Biotech Ltd.). 2.4. Statistical analysis The comparison of frequencies was performed using the Chi-square test, the Fisher’s exact test and the com-

Antibodies to HCV were detected in 3.52% of the leprosy patients (7/199; 4 male and 3 female; mean age of 65 years) and in 0.15% of the controls (1/681; male; 57 years old). The prevalence of HCV infection was significantly higher in the patients when compared to the controls (odds ratio (OR) = 24.79; 95% CI 3.03–202.74; p < 0.001) (Fig. 1). Three of the HCVpositive patients underwent transfusion procedures in the past. Even excluding these patients submitted to transfusion, there was still a significant association between the two infections (odds ratio = 14.17; 95% CI 1.57–127.50). Although all HCV-positive patients present the lepromatous form as shown in Table 2 (n = 7 patients with lepromatous form), no significant difference was found for HCV infection between the distinct clinical forms of the disease (p = 0.640). In addition, an increased risk for HCV infection was observed in institutionalized patients when compared to out-patients (odds ratio = 14.74; 95% CI 1.73–689.74; p = 0.004). Two years after sample collection, one positive patient to hepatitis C died (male, 90 years old). 3.2. Anti-HIV 1 + 2 antibodies There was no difference in the prevalence of HIV infection between leprosy patients and controls (OR = 3.43; 95% CI 0.21–55.16; p = 0.4) (Fig. 1). The only a-HIV-positive patient (female; 41 years old) had the borderline form of leprosy, diagnosed in 2001 and reported to have been contaminated by her husband in 2000; although she might have presented the M. leprae infection previously, but it only had been diagnosed

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Fig. 1. Prevalence rates of anti-HTLV I + II, anti-HIV 1 + 2 and anti-HCV antibodies in leprosy patients and controls from Southern Brazil. Table 2 HCV, HIV1 + 2 and HTLV I + II infection in leprosy patients and healthy controls from Southern Brazil Co-infections

HCV HIV 1 + 2 HTLV I + II 0/0 a b c

Patients

Controls

Sex (M/F)

Assistency form (out-patients/inpatients)

ORa

(95% CI) (lepromatous/others)

Disease form (95% CI)

ORb

4/3 0/1 0/0

1/6 0/1 0/0

14.95 (1.76–127.03) 3.43 (0.21–55.16) 1.14 (0.05–28.02)

7/0 0/1 0/0

7.67 (0.43–136.62) – –

Sex (M/F)

ORc (95% CI)

1/0 1/0 0/1

24.79 (3.03–202.74) – –

Out-patients vs. in-patients. Lepromatous vs. other forms. Patients vs. controls.

after the first symptoms. This patient was admitted to the hospital at the beginning of 2003 with acute respiratory distress and metabolic acidosis, which lead to septic shock and death. 3.3. Anti-HTLV I + II antibodies None of the patients was positive for a-HTLV I + II antibodies (Table 1). One of the controls (female, 52 years old) showed positivity for a-HTLV I + II. These results demonstrated no difference in a-HTLV I + II positivity between patients and controls (OR = 1.14; 95% CI 0.05–28.02; p = 1.000) (Fig. 1). 4. Discussion It has been estimated that 3% of the world population is infected with HCV (Strauss, 2001). According to the National Agency of Health Vigilance, the prevalence of a-HCV in Brazil ranges from 1.0 to 2.5% in the general population and 0.5% in blood donors from South region in 2000. Among leprosy patients, a high prevalence of HCV infection has been reported by different authors around the world (Cisse et al., 1990; Denis et al., 1991, 1994; Frommel et al., 1993; Egawa et al., 1996).

Our data confirmed a high positivity of co-infection by HCV in leprosy patients from Southern Brazil (p < 0.001). However, the high prevalence of a-HCV in leprosy patients reported by some authors (Cisse et al., 1990; Egawa et al., 1996) may be overestimated due to the lack of confirmatory tests of the positive or doubtful samples in EIA. In fact, one study that did not use any confirmatory test presented a rate of 85.4% (Cisse et al., 1990), whereas an a-HCV prevalence of 7.3% using immunoblot as confirmatory test was observed in the same African patients (Denis et al., 1991). Our findings using EIA and immunoblot (3.52%) were similar to the results reported in Ethiopia (3.6%) that in addition to immunoblot, used a dot–blot immunoassay and another EIA to confirm the indeterminate results in immunoblot (Frommel et al., 1993). These data show the necessity of confirmatory tests in the investigation of hepatitis C in order to avoid false positive results. On the other hand, Renaudineau et al. (1996) in Senegal did not show any difference in the prevalence of anti-HCV antibodies comparing leprosy patients with normal population. This could be explained by the fact that the leprosy patients were not confined in a leprosarium and that the authors used a third generation EIA that decreased false positive results.

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Our results differ from the study in Goiˆania in the central region of Brazil (Rosa et al., 1996) that recruited patients in 1991 and did not demonstrate higher risk of HCV infection in leprosy patients. This might be attributed to the different local epidemiology and to the lack of matched controls. Although some studies reveal no difference in the risk to hepatitis C co-infection among the two polar forms of Hansen disease (Egawa et al., 1996; Denis et al., 1994), in our study all positive patients for HCV had the lepromatous form of the disease suggesting that patients with this form are at high risk to develop HCV co-infection (OR = 7.67; 95% CI 0.43–136.36). This study also shows a higher risk to hepatitis C co-infection among institutionalized leprosy patients (OR = 14.95; p = 0.003) confirming data reported by Frommel et al. (1993) in Ethiopia. The higher risk to the HCV co-infection conferred by confinement might be related to the fact that in-patients often have wounds on hands and feet and have undergone surgical procedures, which after blood transfusion and the use of drugs, are considered important risk factors for hepatitis C. These factors in an institutional setting may well contribute to the higher prevalence of HCV in these patients. In addition, all a-HCV positive patients also presented HBV infection. Since the main transmission route of HBV is sexual, followed by the use of drugs, and that the latter is the main pathway of HCV dissemination, there is a possibility that these patients have been infected by the same route. One HCV positive leprosy patient also revealed positivity for syphilis. The high prevalence of co-infections in these patients may be related to the deficient immunity status of lepromatous form and to the fact that the majority (6/7) of them were institutionalized. The only positive patient to HIV 1 + 2 in this study was a woman that at the time of sample collection (09/2002) showed no clinical manifestation of both diseases. After 4 months, this patient, that was under AIDS and leprosy reaction I treatment, was admitted to the hospital presenting acute respiratory distress and metabolic acidosis, probably caused by antiretroviral medication. Unfortunately, she presented septic shock with multi-organs dysfunction and death. According to this evidence, interaction of leprosy with HIV 1 + 2 infection may aggravate the clinical course of both diseases, increasing morbidity and mortality in co-infected patients. It is known that association between HIV and mycobacteria such as M. tuberculosis and M. avium is very strong. Although our data shows no association between HIV and M. leprae, the odds ratio of 3:43 suggests that leprosy patients have higher risk to acquire HIV 1 + 2 infection, which may be due to the particular

cellular mechanisms involved in leprosy pathogenesis. There is the probability that with new epidemiological studies including a larger number of individuals, this data might become statistically significant. Divergent results were observed regarding leprosy and HTLV I + II antibodies. In general, studies in endemic areas of HTLV I + II showed significant association (Hanada et al., 1989; Lechat et al., 1997; Verdier et al., 1990) between the two infections, with exception of some African countries (Verdier et al., 1990). None of the leprosy patients included in this study presented antibodies to HTLV I + II, and only one control revealed positivity to a-HTLV I + II by EIA without the use of confirmatory test. As Brazil is not considered endemic to HTLV I and II infection, the present study confirmed the hypothesis that increased prevalence of HTLV I + II infection occurs in leprosy patients only in endemic regions. In conclusion, our results demonstrate an increased prevalence of HCV infection in Brazilian leprosy patients and that both lepromatous form and confinement can lead to higher risk of HCV infection. These findings indicate the need of special care in monitoring HCV and HIV co-infections in leprosy patients. Acknowledgments We thank Lablife Ltd. for providing all the ELISA kits, and the Serology Lab of Hemotherapy Service from Clinic Hospital of Federal University for their technical support, Dr. Ewalda Von Volsen Stahlke and staff from the Health State Department of Paran´a and Sanitary Dermatology Hospital of Paran´a for their collaboration in the selection of the patients. We thank the Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico – CNPq. References Andrade, V.L., Alves, T.M., Avelleira, J.C.R., Bayona, M., 1997. Prevalence of HIV1 in leprosy patients in Rio de Janeiro, Brazil. Acta Leprol. 10, 159–163. Awofeso, N., 1995. Aids and tuberculosis/leprosy in Nigeria: the urbanization factor. Acta Leprol. 9, 149–151. Blum, L., Ogougbemy, M., M’Boup, S., Grimaud, J., Millan, J., Launois, P., Gaye, Y., Le Guenno, B., Gaye, A., Flageul, B., 1992. Epidemiological study of HIV soroprevalence in a leprosy patient population in Senegal. Acta Leprol. 8, 35–41. Cisse, M.F., Agius, G., Dindinaud, G., Samb, A., Castets, M., 1990. Anti-hepatitis C virus antibodies in Africans with leprosy. Presse Med. 19, 1989. Denis, F.L., Aussel, L., Ranger, S., Martin, P., Itoua-N’Gaporo, A., Frommel, D., Teckle-Haimanot, R.T., Sangare, A., M’Boup, S., Millan, J., Al Qubati, Y., 1994. Prevalence of antibodies to hepatitis

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