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Risk factors for mucosal manifestation of American cutaneous leishmaniasis
Transactions of the Royal Society of Tropical Medicine and Hygiene (2005) 99, 55—61
Risk factors for mucosal manifestation of American cutaneous leishmaniasis George L.L. Machado-Coelhoa,∗, Waleska T. Caiaffab, Odair Genaroc,, Paulo A. Magalh˜ aesd,, Wilson Mayrinkc a
Department of Pharmacy, Federal University of Ouro Preto, Rua Costa Sena 171, 35400-000 Ouro Preto, MG, Brazil b Department of Social and Preventive Medicine of Federal University of Minas Gerais, Belo Horizonte, Brazil c Department of Parasitology of Federal University of Minas Gerais, Belo Horizonte, Brazil d National Health Foundation of Ministry of Health, Caratinga, Brazil Received 29 April 2003 ; received in revised form 18 August 2003; accepted 20 August 2003 KEYWORDS Leishmaniasis; Epidemiology; Risk factors; Mucosal lesions; Brazil
Summary A case-comparison study was carried out to identify risk factors for mucosal manifestations of American cutaneous leishmaniasis (ACL) in southeast Brazil, using a series of 2820 patients, diagnosed with ACL between 1966 and 1999. The significant factors independently associated with mucosal leishmaniasis were: gender, age, nutritional status and length of disease. Mucosal leishmaniasis occurred 1.7 times more frequently among males than females; twice as often in individuals older than 22 years compared with the younger group; almost four times as often in individuals with severe malnutrition compared with those who were well nourished; and almost four times more frequently in individuals reporting the disease for more than 4 months compared with those reporting a shorter duration of the disease. Among individuals older than 22 years the risk of mucosal leishmaniasis increased significantly (from 1.9 to 9.6) as the nutritional status decreased, when compared with younger and well-nourished patients. The characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL. © 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +55 31 3559 1638;
fax: +55 31 3559 1628. E-mail address: [email protected] (G.L.L. Machado-Coelho). Deceased.
1. Introduction American cutaneous leishmaniasis (ACL) is caused by Leishmania parasites of the subgenera Viannia and Leishmania and is endemic in 21 countries
0035-9203/$ — see front matter © 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2003.08.001
56 in the New World where about 39 million individuals are considered to be at risk of acquiring the disease (Ashford et al., 1992). In Brazil, it has been estimated that about 35 000 people were newly diagnosed with ACL annually from 1992 to 2001 (FUNASA, 2003). American cutaneous leishmaniasis is highly endemic in Brazil, and the disease has been reported increasingly in all age and gender groups in some regions (Machado-Coelho et al., 1999; Pereira and Fonseca, 1994). American cutaneous leishmaniasis has three major clinical manifestations in humans: cutaneous leishmaniasis (CL), mucosal leishmaniasis (ML) and diffuse leishmaniasis. Cutaneous leishmaniasis, the most common and least severe form, usually occurs in individuals with an effective immune response. It is characterized by a welldefined ulcer in the skin with frequent findings of amastigotes. Mucosal leishmaniasis presents as a slow but progressive destruction of mucosal and submucosal tissues in the nose and mouth, with rare findings of amastigotes and exaggerated delayed-type hypersensitivity (DTH). Most of the cases are due to infection with Leishmania (Viannia) braziliensis or, less frequently, L. (V.) panamensis and L. (V.) guyanensis (Desjeux, 1996). Diffuse leishmaniasis usually occurs in patients with a poor immune response to Leishmania antigens and is characterized by multiple nodular skin lesions, usually full of parasites. No protective cell-mediated immunity or DTH can be demonstrated in this form (Convit et al., 1993). Clinical and epidemiological evidence suggest that nutritional deficiency leads to increased susceptibility to infection, related to an impaired immunological response (Chandra, 1997; Neumann et al., 1975). Some studies have shown that children are at a greater risk of developing severe visceral leishmaniasis when they are younger and malnourished (Badar´ o et al., 1986; Dye and Williams, 1993). Others have reported that the incidence of CL was similar among protein-energy malnourished and well-nourished rural Ecuadorian children, but higher in children with iron deficiency (Weigel et al., 1995). In an experimental study, protein malnourished mice failed to clear effectively an infection caused by L. mexicana (P´ erez et al., 1979). This study aimed to determine the risk factors associated with severe forms of ACL, defined here as ML, using a very large series of out-patients diagnosed with ACL, either cutaneous or mucosal, in a Brazilian referral treatment clinic, over thirtythree years.
G.L.L. Machado-Coelho et al.
2. Materials and methods 2.1. Study population The study was conducted in Caratinga microregion, an area endemic for leishmaniasis, located in a predominantly agricultural area in Rio Doce Valley, in eastern Minas Gerais State, Southeast Brazil (Machado-Coelho et al., 1999). Information on 2820 patients, newly diagnosed with ACL between 1966 and 1999, was collected through a specific passive surveillance system and included in this casecomparison study. For the past 33 years (1966—1999), medical assistance and surveillance has been provided in this area by the State Health Department in association with the Federal University of Minas Gerais; the latter is a reference centre for leishmaniasis. This study was approved by the Institution Review Board (UFOP CEP number 2002/27).
2.2. Diagnosis Patients with one or more typical active cutaneous lesions were clinically diagnosed with CL. Mucosal leishmaniasis was diagnosed in patients with mucosal lesions, such as naso-buco-pharyngeal lesions, with or without concomitant skin lesions. Every clinical diagnosis was confirmed by a positive Montenegro Skin Test (MST) (Melo et al., 1977) and/or parasitological tests. Parasitological diagnosis was confirmed by the identification of the parasites in tissue biopsy materials, obtained from skin or mucosal ulcers. Imprints of tissue fragments on glass slides were stained with Giemsa and examined microscopically at 1000×.
2.3. Variables A trained data collector extracted data from the medical charts, using a pre-tested and standardized form. Variables extracted included demographic details (age, gender, skin colour and residence), clinical details (clinical form, number of lesions, anatomical site and duration of disease before diagnosis), anthropometrics (weight and height) and diagnostics (parasitological and skin test results). Severe and moderate protein-energy malnutrition was defined using two measures depending upon the age of the patient. Body Mass Index (BMI) was used for those ≥18 years old and Z-score of height/age index for younger patients. Stratification by gender was done using a BMI cut-off less than 18 and 16 kg/m2 for moderate and severe among
Risk factors for mucosal manifestation men and less than 17 and 15 kg/m2 for women respectively (WHO, 1985). For the Z-score criteria, severe, moderate and light chronic protein-energy malnutrition was defined by the cut off of −3, −2 and −1.28 respectively (Goulart, 1997).
2.4. Data analysis Frequency distributions were generated separately for each variable and the median was calculated to define the categorical variables. The CL and ML cases were cross-classified with demographic, clinical, nutritional and diagnostic variables to generate odds ratios (OR) with 95% confidence intervals (CI) to guide interpretation. Variables, either biologically plausible or statistically significant in univariate analysis, were examined simultaneously using multiple logistic regression (Kleinbaum et al., 1998).
3. Results A total of 2820 ACL patients were identified between 1966 and 1999; 94.9% were diagnosed with CL and 5.1% with ML (Table 1). The median age was 22 years (range from 4 months to 89 years); ML patients (31 years) were older than CL patients (21 years) (P < 0.001). Also, ML occurred more frequently among males (66.9%) than females (P < 0.01). There was no significant difference between CL and ML patients regarding residence (urban/rural) or skin colour (black/non black). Light, moderate, and severe malnutrition were observed at rates of 44.3, 22.4 and 3.6% respec-
57 tively for patients aged 18 years or more. For comparison, light/moderate and severe malnutrition were less frequent among patients younger than 18 years: 6 and 0.9% respectively. Mucosal leishmaniasis and CL occurred at similar rates among wellfed and moderately malnourished patients. However, patients with ML presented 3.4 times (95% CI: 1.5—7.7) more chance of being malnourished (5.6%) than patients with CL (1.7%) (Table 1). The number of lesions varied from 1 to 36; 77% of patients presented only one lesion. Lesions were more frequently observed on the legs (44%), and less frequently on the palate (0.04%) and oropharyngeal sites (0.2%) (Figure 1). The distribution of lesions did not differ according to clinical presentation (Table 1). Duration of the disease, defined as the time interval reported between the initial observation of the lesion and clinical diagnosis, varied from 4 days to 44 years with a median of 2 months. Duration of the disease was similar for men and women (60 days, P = 0.53), but differed significantly when stratified by clinical presentation, being longer in patients with ML than with CL, with an average duration of 120 and 60 days respectively (P < 0.001). Also, duration of the disease was significantly longer for individuals older than 22 years as compared with the younger group (P = 0.045). The significant demographic and clinical variables in the univariate analysis were used to develop the multiple logistic regression model, adjusted by calendar year, as shown in Table 2. Mucosal leishmaniasis occurred 1.7 times more frequently in males than females (95% CI: 1.1—2.5), twice as often in individuals aged over 22 years
Table 1 Univariate associations of demographic and clinic variables of 2820 cases of American cutaneous leishmaniasis Characteristics Demographics Age (>22 years) Male gender Non-black skin colour Rural residence
Mucosal lesion No. patientsa (%)
Cutaneous lesion No. patientsa (%)
Odds ratio (CI 95%)
89 97 126 117
1286 1503 2337 2204
1.7 1.6 0.9 0.9
(61.4) (66.9) (87.5) (80.7)
(48.2) (56.2) (88.1) (82.4)
Pb
(1.2—2.4) (1.1—2.3) (0.6—1.6) (0.6—1.4)
0.002 0.01 0.82 0.60
Nutritional status Light/moderate malnutrition Severe malnutrition
25 (20.0) 7 (5.6)
455 (19.2) 41 (1.7)
1.0 (0.7—1.7) 3.4 (1.5—7.7)
0.83 0.002
Clinical Number of lesions (>1) Number of locations (>1) Time of disease (>4 months)
38 (26.6) 13 (9.4) 68 (47.2)
607 (22.8) 143 (5.6) 471 (17.8)
1.2 (0.8—1.8) 1.7 (1.0—3.2) 4.1 (2.9—5.8)
0.29 0.06 <0.001
CI: Confidence interval. a Actual n may vary because of missing data (mucosal lesion = 145, cutaneous lesion = 2675). b P value obtained by statistics or exact probabilities.
58
G.L.L. Machado-Coelho et al.
Figure 1. Site of the lesions for 2820 patients with American cutaneous leishmaniasis.
than in the younger group (95% CI: 1.3—3.1), four times more frequently in those with clinical malnutrition than in the well-nourished individuals (95% CI: 1.6—9.5), and four times more frequently in those reporting more than 4 months duration of the disease (95% CI: 2.7—5.7). The risk of ML as compared to CL in patients with light or moderate malnutrition remained non-significant (OR =
1.4, 95% CI: 0.8—2.4) as in the previous univariate analysis. Table 3 shows the risk modification analysis, adjusted by gender and calendar year, for ML regarding age and nutritional state. Considering the younger (22 years old or less) and well-nourished individuals as a reference group, the risk of presenting ML was three times more (95% CI: 0.9—9.2)
Table 2 Univariate and main effects of multiple logistic regression models for 145 cases with mucosal lesion and 2675 cases with cutaneous lesion of American cutaneous leishmaniasis Variables
Crude odds ratio (95% CI)
Male gender Age >22 years Light or moderate malnutrition Severe malnutrition Disease evolution time (>4 months)
1.6 1.7 1.0 3.4 4.1
b
(1.1—2.3) (1.2—2.4)b (0.7—1.7) (1.5—7.7)b (2.9—5.8)b
Adjusted odds ratioa (95% CI) 1.7 2.0 1.4 3.9 3.9
(1.1—2.5)a (1.3—3.1)a (0.8—2.4) (1.6—9.5)a (2.7—5.7)a
CI: Confidence interval. Missing: 20 cases with mucosal lesion and 321 cases with cutaneous lesion. a Adjusted for the other variables presented for each model and calendar year. b P < 0.01.
Table 3 Bivariate associations of age (years) and protein-energy malnutrition of 2820 cases of American cutaneous leishmaniasis Characteristics
Mucosal lesion
Cutaneous lesion
Age
Malnutrition
No. patients (%)
No. patients (%)
≤22 ≤22 ≤22 >22 >22 >22
No Light/moderate Severe No Light/moderate Severe
25 14 4 68 11 3
773 404 33 1100 51 8
(3.1) (3.3) (10.8) (5.8) (17.7) (27.3)
CI: Confidence interval. a Adjusted by sex and calendar year.
(96.9) (96.7) (89.2) (94.2) (82.3) (72.7)
Odds ratio (CI 95%)
Adjusted odds ratioa (CI 95%)
1.0 1.1 3.8 1.9 6.7 11.6
1.0 0.9 3.0 1.9 5.8 9.6
(0.5—2.2) (1.0—12.2) (1.2—3.1) (2.9—15.1) (2.3—52.4)
(0.5—1.8) (1.0—9.2) (1.2—3.0) (2.7—12.5) (2.4—39.0)
Risk factors for mucosal manifestation
Table 4
59
Bivariate associations of time of disease and age of 2820 cases of American cutaneous leishmaniasis
Characteristics
Mucosal lesion
Cutaneous lesion
Time of disease (months)
Age (years)
No. patients (%)
No. patients (%)
≤4 ≤4 >4 >4
≤22 >22 ≤22 >22
41 35 14 54
1145 1028 225 245
(3.5) (3.3) (5.9) (18.1)
(96.5) (96.7) (94.1) (81.9)
Odds ratio (CI 95%)
Adjusted odds ratioa (CI 95%)
1.0 1.0 (0.6—1.5) 1.7 (0.9—3.4) 6.16 (3.9—9.7)
1.0 1.0 (0.6—1.5) 1.7 (0.9—3.1) 5.7 (3.7—8.8)
CI: Confidence interval. a Adjusted by sex and calendar year.
in the younger individuals with serious malnutrition, two times (95% CI: 1.2—2.9) more in the wellnourished individuals over 22 years, six times more (95% CI: 2.7—12.5) in individuals over 22 years with light or moderate malnutrition and ten times more (95% CI: 2.4—38.9) in individuals over 22 years with serious malnutrition. No differences in risk were observed among the young individuals with light or moderate malnutrition in relation to the reference group (OR = 0.9, 95% CI: 0.5—1.8). It is important to point out that, even taking into account confounders in the adjusted model, the modification effect (or synergism) between age and nutritional status was significant among those older than 22 years of age, with a dose-response gradient from two when there was no nutritional deficiency to five up to ten when a degree of nutritional deficiency was considered. Table 4 shows the risk modification analysis, adjusted by gender and calendar year, of ML regarding patients’ age and reported duration of the disease. Considering young individuals (22 years old or less) reporting the disease for less than four months as a reference group, the risk of presenting ML was six times higher (95% CI: 3.7—8.8) in individuals aged over 22 years, reporting the disease for more than four months. No significant differences were observed in the risk among younger individuals reporting more than four months of the disease (OR = 1.7, 95% CI: 0.8—3.1) and among individuals older than 22 years and with four or more months of the disease (OR = 0.97, 95% CI: 0.6—1.5).
4. Discussion We studied 2820 cases of ACL that were diagnosed between 1966 and 1999. About 2675 individuals were reported with CL and 145 with ML, a more severe clinical manifestation. Individuals aged over 22 years, male, severely malnourished and sick for more than four months had a higher risk of present-
ing with ML. A significant gradient of risk relating age and malnutrition was observed. Among patients older than 22 years the risk of a severe manifestation increased as the nutritional status decreased. The increased risk for the more serious clinical forms of leshmaniasis associated with the degree of malnutrition has been observed in a few clinical and epidemiological studies. In Ecuador, children with simultaneous CL and chronic protein-energy malnutrition presented a more severe clinical manifestation of the disease (Weigel et al., 1995). Clinical findings may corroborate epidemiological studies showing that malnourished patients presenting with subclinical, active or even secondary infections were more susceptible to activation or reactivation, with a spread of Leishmania from the skin to the mucosal membranes (Saravia et al., 1985). The association with malnourishment has also been reported in an animal model. Mice infected by L. mexicana and fed on a poor protein and vitamin diet presented a larger risk of developing cutaneous lesions than those with a normal protein and vitamin intake (P´ erez et al., 1979). In another experiment, the Th1 type immune response decreased during an L. major infection (Taylor et al., 1996). Additionally, the association between malnutrition and age as risk factors for severe forms of visceral leishmaniasis is well known, in both clinical and epidemiological studies (Badar´ o et al., 1986; Dye and Williams, 1993). An association between age and severe forms of ACL has been observed by other authors (Avila et al., 1992; Marsden, 1986). Mucosal leishmaniasis was more frequent in patients infected by L. braziliensis and usually happens in two stages after the bite of an infected sandfly: a primary cutaneous lesion followed by a secondary involvement of the mucosal layer (Marsden, 1986). Also, A. Volpini (personal communication) identified Leishmania species in histological samples; PCR-Restriction Fragment Length Polymorphism has shown that L. braziliensis was the sole species in Caratinga. Cutaneous lesions may or may not heal
60 spontaneously, but a small proportion may be followed by metastatic mucosal lesions, after months, years or decades (Barreto et al., 1981; Marsden et al., 1984). In this study, severe forms were associated with a longer duration of the disease, suggesting a longer incubation period. Similar results were observed by other authors, who reported that multiple or extensive cutaneous lesions of long duration are more frequently associated with mucosal complications, determining ML (Llanos-Cuentas et al., 1984). Regarding gender differences, in an endemic area of ACL, mucosal forms have been described more frequently in men (Marsden et al., 1984). Some studies have hypothesized that the gender difference observed in some parasitic disease can be attributed to hormonal effects. However, controversy still exists regarding the role of sex hormones in the cellular immune response (Brabin and Brabin, 1992; Bundy, 1988). Although it is believed that sex hormones may influence the establishment and the course of parasitic diseases, behavioural factors, making male individuals more likely to be exposed to vectors in forests and other transmission environments, are probably equally or more important, mainly when considering the peculiar epidemiological patterns characterized by extraforestal transmission described in Brazil (Forattini et al., 1976; Mayrink et al., 1979). Therefore, the association between male gender and ML probably reflects either the higher frequency of Leishmania infection or of CL observed in men (Armijos et al., 1997; Dedet et al., 1989). Another explanation for this gender differential could be related to the reluctance of adult males to seek medical aid in the initial period of the disease, leading to prolonged infection and exacerbation of the disease. However, in this temporal series, the median disease duration was similar in males and females, independently of age and clinical form of leishmaniasis, thus not supporting this argument. Lesions were concentrated on the upper and lower limbs. A higher frequency of ML was observed among those patients with a larger number of lesions and more anatomical sites involved, although this difference was not significant. Similar results were observed by other authors (Maigon et al., 1994; Marsden et al., 1984). Before drawing any conclusion, the several limitations of this study should be acknowledged. The data used in this study were not collected for an aetiologic investigation. Therefore, several risk factors that could be related to the severity of the disease could not be ascertained, giving the possibility of the presence of residual confounders. In addition, various biases could have influenced the
G.L.L. Machado-Coelho et al. frequency of cases because the study was retrospective. Memory and surveillance bias do not seem to have occurred in the measurement of clinical forms because the medical-sanitary team and the procedures for diagnosis had not changed over the 33 years covered by the study. However, the evaluation of risk factors using passive detection of cases is subject to selection bias. To avoid such bias, this analysis refers only to the patients who had their residence in a selected municipality for which the health centre was a referral centre for ACL diagnosis and treatment. The determination of a causal association among risk factors susceptible to alteration by the disease itself, such as the association between the protein-energy malnutrition and ACL in this present study, is subject to an inherent temporal bias. Inspecting the data in this study we observed that the majority of patients diagnosed with ML had their lesions in the nasal septa, with a very low probability of presenting feeding problems which in turn could compromise their nutritional status. Also, out of 11 patients with lesions located in the palate or oropharynx, which potentially could interfere with the ingestion, only three were undernourished. Consequently, it seems reasonable that malnourishment was present before the infection. Nonetheless, the only way to revolve this bias would be to design a prospective study. However, because of the rarity of the severe form of leishmaniasis only collection of cases over a very long time period would make possible a powerful study. Finally, considering the complexity of the disease and the need for prompt diagnosis and treatment, the characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL (Gordis, 2000). However, because of the severity of the disease and all the difficulties observed during the treatment of leishmaniasis with drugs, these studies should be validated through other population-based studies. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgments Our thanks to Jair Cec´ılio de Paula for your technical support during the last 33 years and to Jane de Paula Medina and Edna Cristina Rocha Muniz for data revision. This work was supported by the CNPq
Risk factors for mucosal manifestation (financial support and salary grant), BIOBRAS, Capes (grant), PRPq/UFMG.
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Risk factors for mucosal manifestation of American cutaneous leishmaniasis George L.L. Machado-Coelhoa,∗, Waleska T. Caiaffab, Odair Genaroc,, Paulo A. Magalh˜ aesd,, Wilson Mayrinkc a
Department of Pharmacy, Federal University of Ouro Preto, Rua Costa Sena 171, 35400-000 Ouro Preto, MG, Brazil b Department of Social and Preventive Medicine of Federal University of Minas Gerais, Belo Horizonte, Brazil c Department of Parasitology of Federal University of Minas Gerais, Belo Horizonte, Brazil d National Health Foundation of Ministry of Health, Caratinga, Brazil Received 29 April 2003 ; received in revised form 18 August 2003; accepted 20 August 2003 KEYWORDS Leishmaniasis; Epidemiology; Risk factors; Mucosal lesions; Brazil
Summary A case-comparison study was carried out to identify risk factors for mucosal manifestations of American cutaneous leishmaniasis (ACL) in southeast Brazil, using a series of 2820 patients, diagnosed with ACL between 1966 and 1999. The significant factors independently associated with mucosal leishmaniasis were: gender, age, nutritional status and length of disease. Mucosal leishmaniasis occurred 1.7 times more frequently among males than females; twice as often in individuals older than 22 years compared with the younger group; almost four times as often in individuals with severe malnutrition compared with those who were well nourished; and almost four times more frequently in individuals reporting the disease for more than 4 months compared with those reporting a shorter duration of the disease. Among individuals older than 22 years the risk of mucosal leishmaniasis increased significantly (from 1.9 to 9.6) as the nutritional status decreased, when compared with younger and well-nourished patients. The characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL. © 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +55 31 3559 1638;
fax: +55 31 3559 1628. E-mail address: [email protected] (G.L.L. Machado-Coelho). Deceased.
1. Introduction American cutaneous leishmaniasis (ACL) is caused by Leishmania parasites of the subgenera Viannia and Leishmania and is endemic in 21 countries
0035-9203/$ — see front matter © 2004 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.trstmh.2003.08.001
56 in the New World where about 39 million individuals are considered to be at risk of acquiring the disease (Ashford et al., 1992). In Brazil, it has been estimated that about 35 000 people were newly diagnosed with ACL annually from 1992 to 2001 (FUNASA, 2003). American cutaneous leishmaniasis is highly endemic in Brazil, and the disease has been reported increasingly in all age and gender groups in some regions (Machado-Coelho et al., 1999; Pereira and Fonseca, 1994). American cutaneous leishmaniasis has three major clinical manifestations in humans: cutaneous leishmaniasis (CL), mucosal leishmaniasis (ML) and diffuse leishmaniasis. Cutaneous leishmaniasis, the most common and least severe form, usually occurs in individuals with an effective immune response. It is characterized by a welldefined ulcer in the skin with frequent findings of amastigotes. Mucosal leishmaniasis presents as a slow but progressive destruction of mucosal and submucosal tissues in the nose and mouth, with rare findings of amastigotes and exaggerated delayed-type hypersensitivity (DTH). Most of the cases are due to infection with Leishmania (Viannia) braziliensis or, less frequently, L. (V.) panamensis and L. (V.) guyanensis (Desjeux, 1996). Diffuse leishmaniasis usually occurs in patients with a poor immune response to Leishmania antigens and is characterized by multiple nodular skin lesions, usually full of parasites. No protective cell-mediated immunity or DTH can be demonstrated in this form (Convit et al., 1993). Clinical and epidemiological evidence suggest that nutritional deficiency leads to increased susceptibility to infection, related to an impaired immunological response (Chandra, 1997; Neumann et al., 1975). Some studies have shown that children are at a greater risk of developing severe visceral leishmaniasis when they are younger and malnourished (Badar´ o et al., 1986; Dye and Williams, 1993). Others have reported that the incidence of CL was similar among protein-energy malnourished and well-nourished rural Ecuadorian children, but higher in children with iron deficiency (Weigel et al., 1995). In an experimental study, protein malnourished mice failed to clear effectively an infection caused by L. mexicana (P´ erez et al., 1979). This study aimed to determine the risk factors associated with severe forms of ACL, defined here as ML, using a very large series of out-patients diagnosed with ACL, either cutaneous or mucosal, in a Brazilian referral treatment clinic, over thirtythree years.
G.L.L. Machado-Coelho et al.
2. Materials and methods 2.1. Study population The study was conducted in Caratinga microregion, an area endemic for leishmaniasis, located in a predominantly agricultural area in Rio Doce Valley, in eastern Minas Gerais State, Southeast Brazil (Machado-Coelho et al., 1999). Information on 2820 patients, newly diagnosed with ACL between 1966 and 1999, was collected through a specific passive surveillance system and included in this casecomparison study. For the past 33 years (1966—1999), medical assistance and surveillance has been provided in this area by the State Health Department in association with the Federal University of Minas Gerais; the latter is a reference centre for leishmaniasis. This study was approved by the Institution Review Board (UFOP CEP number 2002/27).
2.2. Diagnosis Patients with one or more typical active cutaneous lesions were clinically diagnosed with CL. Mucosal leishmaniasis was diagnosed in patients with mucosal lesions, such as naso-buco-pharyngeal lesions, with or without concomitant skin lesions. Every clinical diagnosis was confirmed by a positive Montenegro Skin Test (MST) (Melo et al., 1977) and/or parasitological tests. Parasitological diagnosis was confirmed by the identification of the parasites in tissue biopsy materials, obtained from skin or mucosal ulcers. Imprints of tissue fragments on glass slides were stained with Giemsa and examined microscopically at 1000×.
2.3. Variables A trained data collector extracted data from the medical charts, using a pre-tested and standardized form. Variables extracted included demographic details (age, gender, skin colour and residence), clinical details (clinical form, number of lesions, anatomical site and duration of disease before diagnosis), anthropometrics (weight and height) and diagnostics (parasitological and skin test results). Severe and moderate protein-energy malnutrition was defined using two measures depending upon the age of the patient. Body Mass Index (BMI) was used for those ≥18 years old and Z-score of height/age index for younger patients. Stratification by gender was done using a BMI cut-off less than 18 and 16 kg/m2 for moderate and severe among
Risk factors for mucosal manifestation men and less than 17 and 15 kg/m2 for women respectively (WHO, 1985). For the Z-score criteria, severe, moderate and light chronic protein-energy malnutrition was defined by the cut off of −3, −2 and −1.28 respectively (Goulart, 1997).
2.4. Data analysis Frequency distributions were generated separately for each variable and the median was calculated to define the categorical variables. The CL and ML cases were cross-classified with demographic, clinical, nutritional and diagnostic variables to generate odds ratios (OR) with 95% confidence intervals (CI) to guide interpretation. Variables, either biologically plausible or statistically significant in univariate analysis, were examined simultaneously using multiple logistic regression (Kleinbaum et al., 1998).
3. Results A total of 2820 ACL patients were identified between 1966 and 1999; 94.9% were diagnosed with CL and 5.1% with ML (Table 1). The median age was 22 years (range from 4 months to 89 years); ML patients (31 years) were older than CL patients (21 years) (P < 0.001). Also, ML occurred more frequently among males (66.9%) than females (P < 0.01). There was no significant difference between CL and ML patients regarding residence (urban/rural) or skin colour (black/non black). Light, moderate, and severe malnutrition were observed at rates of 44.3, 22.4 and 3.6% respec-
57 tively for patients aged 18 years or more. For comparison, light/moderate and severe malnutrition were less frequent among patients younger than 18 years: 6 and 0.9% respectively. Mucosal leishmaniasis and CL occurred at similar rates among wellfed and moderately malnourished patients. However, patients with ML presented 3.4 times (95% CI: 1.5—7.7) more chance of being malnourished (5.6%) than patients with CL (1.7%) (Table 1). The number of lesions varied from 1 to 36; 77% of patients presented only one lesion. Lesions were more frequently observed on the legs (44%), and less frequently on the palate (0.04%) and oropharyngeal sites (0.2%) (Figure 1). The distribution of lesions did not differ according to clinical presentation (Table 1). Duration of the disease, defined as the time interval reported between the initial observation of the lesion and clinical diagnosis, varied from 4 days to 44 years with a median of 2 months. Duration of the disease was similar for men and women (60 days, P = 0.53), but differed significantly when stratified by clinical presentation, being longer in patients with ML than with CL, with an average duration of 120 and 60 days respectively (P < 0.001). Also, duration of the disease was significantly longer for individuals older than 22 years as compared with the younger group (P = 0.045). The significant demographic and clinical variables in the univariate analysis were used to develop the multiple logistic regression model, adjusted by calendar year, as shown in Table 2. Mucosal leishmaniasis occurred 1.7 times more frequently in males than females (95% CI: 1.1—2.5), twice as often in individuals aged over 22 years
Table 1 Univariate associations of demographic and clinic variables of 2820 cases of American cutaneous leishmaniasis Characteristics Demographics Age (>22 years) Male gender Non-black skin colour Rural residence
Mucosal lesion No. patientsa (%)
Cutaneous lesion No. patientsa (%)
Odds ratio (CI 95%)
89 97 126 117
1286 1503 2337 2204
1.7 1.6 0.9 0.9
(61.4) (66.9) (87.5) (80.7)
(48.2) (56.2) (88.1) (82.4)
Pb
(1.2—2.4) (1.1—2.3) (0.6—1.6) (0.6—1.4)
0.002 0.01 0.82 0.60
Nutritional status Light/moderate malnutrition Severe malnutrition
25 (20.0) 7 (5.6)
455 (19.2) 41 (1.7)
1.0 (0.7—1.7) 3.4 (1.5—7.7)
0.83 0.002
Clinical Number of lesions (>1) Number of locations (>1) Time of disease (>4 months)
38 (26.6) 13 (9.4) 68 (47.2)
607 (22.8) 143 (5.6) 471 (17.8)
1.2 (0.8—1.8) 1.7 (1.0—3.2) 4.1 (2.9—5.8)
0.29 0.06 <0.001
CI: Confidence interval. a Actual n may vary because of missing data (mucosal lesion = 145, cutaneous lesion = 2675). b P value obtained by statistics or exact probabilities.
58
G.L.L. Machado-Coelho et al.
Figure 1. Site of the lesions for 2820 patients with American cutaneous leishmaniasis.
than in the younger group (95% CI: 1.3—3.1), four times more frequently in those with clinical malnutrition than in the well-nourished individuals (95% CI: 1.6—9.5), and four times more frequently in those reporting more than 4 months duration of the disease (95% CI: 2.7—5.7). The risk of ML as compared to CL in patients with light or moderate malnutrition remained non-significant (OR =
1.4, 95% CI: 0.8—2.4) as in the previous univariate analysis. Table 3 shows the risk modification analysis, adjusted by gender and calendar year, for ML regarding age and nutritional state. Considering the younger (22 years old or less) and well-nourished individuals as a reference group, the risk of presenting ML was three times more (95% CI: 0.9—9.2)
Table 2 Univariate and main effects of multiple logistic regression models for 145 cases with mucosal lesion and 2675 cases with cutaneous lesion of American cutaneous leishmaniasis Variables
Crude odds ratio (95% CI)
Male gender Age >22 years Light or moderate malnutrition Severe malnutrition Disease evolution time (>4 months)
1.6 1.7 1.0 3.4 4.1
b
(1.1—2.3) (1.2—2.4)b (0.7—1.7) (1.5—7.7)b (2.9—5.8)b
Adjusted odds ratioa (95% CI) 1.7 2.0 1.4 3.9 3.9
(1.1—2.5)a (1.3—3.1)a (0.8—2.4) (1.6—9.5)a (2.7—5.7)a
CI: Confidence interval. Missing: 20 cases with mucosal lesion and 321 cases with cutaneous lesion. a Adjusted for the other variables presented for each model and calendar year. b P < 0.01.
Table 3 Bivariate associations of age (years) and protein-energy malnutrition of 2820 cases of American cutaneous leishmaniasis Characteristics
Mucosal lesion
Cutaneous lesion
Age
Malnutrition
No. patients (%)
No. patients (%)
≤22 ≤22 ≤22 >22 >22 >22
No Light/moderate Severe No Light/moderate Severe
25 14 4 68 11 3
773 404 33 1100 51 8
(3.1) (3.3) (10.8) (5.8) (17.7) (27.3)
CI: Confidence interval. a Adjusted by sex and calendar year.
(96.9) (96.7) (89.2) (94.2) (82.3) (72.7)
Odds ratio (CI 95%)
Adjusted odds ratioa (CI 95%)
1.0 1.1 3.8 1.9 6.7 11.6
1.0 0.9 3.0 1.9 5.8 9.6
(0.5—2.2) (1.0—12.2) (1.2—3.1) (2.9—15.1) (2.3—52.4)
(0.5—1.8) (1.0—9.2) (1.2—3.0) (2.7—12.5) (2.4—39.0)
Risk factors for mucosal manifestation
Table 4
59
Bivariate associations of time of disease and age of 2820 cases of American cutaneous leishmaniasis
Characteristics
Mucosal lesion
Cutaneous lesion
Time of disease (months)
Age (years)
No. patients (%)
No. patients (%)
≤4 ≤4 >4 >4
≤22 >22 ≤22 >22
41 35 14 54
1145 1028 225 245
(3.5) (3.3) (5.9) (18.1)
(96.5) (96.7) (94.1) (81.9)
Odds ratio (CI 95%)
Adjusted odds ratioa (CI 95%)
1.0 1.0 (0.6—1.5) 1.7 (0.9—3.4) 6.16 (3.9—9.7)
1.0 1.0 (0.6—1.5) 1.7 (0.9—3.1) 5.7 (3.7—8.8)
CI: Confidence interval. a Adjusted by sex and calendar year.
in the younger individuals with serious malnutrition, two times (95% CI: 1.2—2.9) more in the wellnourished individuals over 22 years, six times more (95% CI: 2.7—12.5) in individuals over 22 years with light or moderate malnutrition and ten times more (95% CI: 2.4—38.9) in individuals over 22 years with serious malnutrition. No differences in risk were observed among the young individuals with light or moderate malnutrition in relation to the reference group (OR = 0.9, 95% CI: 0.5—1.8). It is important to point out that, even taking into account confounders in the adjusted model, the modification effect (or synergism) between age and nutritional status was significant among those older than 22 years of age, with a dose-response gradient from two when there was no nutritional deficiency to five up to ten when a degree of nutritional deficiency was considered. Table 4 shows the risk modification analysis, adjusted by gender and calendar year, of ML regarding patients’ age and reported duration of the disease. Considering young individuals (22 years old or less) reporting the disease for less than four months as a reference group, the risk of presenting ML was six times higher (95% CI: 3.7—8.8) in individuals aged over 22 years, reporting the disease for more than four months. No significant differences were observed in the risk among younger individuals reporting more than four months of the disease (OR = 1.7, 95% CI: 0.8—3.1) and among individuals older than 22 years and with four or more months of the disease (OR = 0.97, 95% CI: 0.6—1.5).
4. Discussion We studied 2820 cases of ACL that were diagnosed between 1966 and 1999. About 2675 individuals were reported with CL and 145 with ML, a more severe clinical manifestation. Individuals aged over 22 years, male, severely malnourished and sick for more than four months had a higher risk of present-
ing with ML. A significant gradient of risk relating age and malnutrition was observed. Among patients older than 22 years the risk of a severe manifestation increased as the nutritional status decreased. The increased risk for the more serious clinical forms of leshmaniasis associated with the degree of malnutrition has been observed in a few clinical and epidemiological studies. In Ecuador, children with simultaneous CL and chronic protein-energy malnutrition presented a more severe clinical manifestation of the disease (Weigel et al., 1995). Clinical findings may corroborate epidemiological studies showing that malnourished patients presenting with subclinical, active or even secondary infections were more susceptible to activation or reactivation, with a spread of Leishmania from the skin to the mucosal membranes (Saravia et al., 1985). The association with malnourishment has also been reported in an animal model. Mice infected by L. mexicana and fed on a poor protein and vitamin diet presented a larger risk of developing cutaneous lesions than those with a normal protein and vitamin intake (P´ erez et al., 1979). In another experiment, the Th1 type immune response decreased during an L. major infection (Taylor et al., 1996). Additionally, the association between malnutrition and age as risk factors for severe forms of visceral leishmaniasis is well known, in both clinical and epidemiological studies (Badar´ o et al., 1986; Dye and Williams, 1993). An association between age and severe forms of ACL has been observed by other authors (Avila et al., 1992; Marsden, 1986). Mucosal leishmaniasis was more frequent in patients infected by L. braziliensis and usually happens in two stages after the bite of an infected sandfly: a primary cutaneous lesion followed by a secondary involvement of the mucosal layer (Marsden, 1986). Also, A. Volpini (personal communication) identified Leishmania species in histological samples; PCR-Restriction Fragment Length Polymorphism has shown that L. braziliensis was the sole species in Caratinga. Cutaneous lesions may or may not heal
60 spontaneously, but a small proportion may be followed by metastatic mucosal lesions, after months, years or decades (Barreto et al., 1981; Marsden et al., 1984). In this study, severe forms were associated with a longer duration of the disease, suggesting a longer incubation period. Similar results were observed by other authors, who reported that multiple or extensive cutaneous lesions of long duration are more frequently associated with mucosal complications, determining ML (Llanos-Cuentas et al., 1984). Regarding gender differences, in an endemic area of ACL, mucosal forms have been described more frequently in men (Marsden et al., 1984). Some studies have hypothesized that the gender difference observed in some parasitic disease can be attributed to hormonal effects. However, controversy still exists regarding the role of sex hormones in the cellular immune response (Brabin and Brabin, 1992; Bundy, 1988). Although it is believed that sex hormones may influence the establishment and the course of parasitic diseases, behavioural factors, making male individuals more likely to be exposed to vectors in forests and other transmission environments, are probably equally or more important, mainly when considering the peculiar epidemiological patterns characterized by extraforestal transmission described in Brazil (Forattini et al., 1976; Mayrink et al., 1979). Therefore, the association between male gender and ML probably reflects either the higher frequency of Leishmania infection or of CL observed in men (Armijos et al., 1997; Dedet et al., 1989). Another explanation for this gender differential could be related to the reluctance of adult males to seek medical aid in the initial period of the disease, leading to prolonged infection and exacerbation of the disease. However, in this temporal series, the median disease duration was similar in males and females, independently of age and clinical form of leishmaniasis, thus not supporting this argument. Lesions were concentrated on the upper and lower limbs. A higher frequency of ML was observed among those patients with a larger number of lesions and more anatomical sites involved, although this difference was not significant. Similar results were observed by other authors (Maigon et al., 1994; Marsden et al., 1984). Before drawing any conclusion, the several limitations of this study should be acknowledged. The data used in this study were not collected for an aetiologic investigation. Therefore, several risk factors that could be related to the severity of the disease could not be ascertained, giving the possibility of the presence of residual confounders. In addition, various biases could have influenced the
G.L.L. Machado-Coelho et al. frequency of cases because the study was retrospective. Memory and surveillance bias do not seem to have occurred in the measurement of clinical forms because the medical-sanitary team and the procedures for diagnosis had not changed over the 33 years covered by the study. However, the evaluation of risk factors using passive detection of cases is subject to selection bias. To avoid such bias, this analysis refers only to the patients who had their residence in a selected municipality for which the health centre was a referral centre for ACL diagnosis and treatment. The determination of a causal association among risk factors susceptible to alteration by the disease itself, such as the association between the protein-energy malnutrition and ACL in this present study, is subject to an inherent temporal bias. Inspecting the data in this study we observed that the majority of patients diagnosed with ML had their lesions in the nasal septa, with a very low probability of presenting feeding problems which in turn could compromise their nutritional status. Also, out of 11 patients with lesions located in the palate or oropharynx, which potentially could interfere with the ingestion, only three were undernourished. Consequently, it seems reasonable that malnourishment was present before the infection. Nonetheless, the only way to revolve this bias would be to design a prospective study. However, because of the rarity of the severe form of leishmaniasis only collection of cases over a very long time period would make possible a powerful study. Finally, considering the complexity of the disease and the need for prompt diagnosis and treatment, the characteristics herein described and correlated with severe forms could be used as diagnostic markers as part of clinical screening in areas endemic for ACL (Gordis, 2000). However, because of the severity of the disease and all the difficulties observed during the treatment of leishmaniasis with drugs, these studies should be validated through other population-based studies. Conflicts of interest statement The authors have no conflicts of interest concerning the work reported in this paper.
Acknowledgments Our thanks to Jair Cec´ılio de Paula for your technical support during the last 33 years and to Jane de Paula Medina and Edna Cristina Rocha Muniz for data revision. This work was supported by the CNPq
Risk factors for mucosal manifestation (financial support and salary grant), BIOBRAS, Capes (grant), PRPq/UFMG.
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