SciELO - Scientific Electronic Library Online

 
vol.60Evaluation of a new brand of immunochromatographic test for visceral leishmaniasis in Brazil made available from 2018Preliminary report of body lice infesting homeless people in Brazil índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

Compartir


Revista do Instituto de Medicina Tropical de São Paulo

versión On-line ISSN 1678-9946

Rev. Inst. Med. trop. S. Paulo vol.60  São Paulo  2018  Epub 13-Sep-2018

http://dx.doi.org/10.1590/s1678-9946201860050 

Brief Communication

Potential role of zinc in the visceromegaly regression and recovery of hematological parameters during treatment of visceral leishmaniasis in children from an endemic area

Débora Cardozo Bonfim Carbone1  2  3 

Lourdes Zélia Garcia Zanoni1  3 

Fernanda Zanoni Cônsolo1 

Simone Camargo Sanches4 

Vanessa Quadros dos Reis3 

Karla de Toledo Candido Muller2 

Cristiano Marcelo Espinola Carvalho2 

Maria Cláudia Silva2 

1Universidade Federal do Mato Grosso do Sul, Programa de Pós-Graduação em Saúde e Desenvolvimento, Campo Grande, Mato Grosso do Sul, Brazil

2Universidade Católica Dom Bosco, Programa de Pós-Graduação em Biotecnologia, Campo Grande, Mato Grosso do Sul, Brazil

3Universidade Federal do Mato Grosso do Sul, Hospital Universitário Maria Aparecida Pedrossian, Campo Grande, Mato Grosso do Sul, Brazil

4Universidade Federal do Mato Grosso do Sul, Rede Centro-Oeste de Pós-Graduação, Pesquisa e Inovação, Campo Grande, Mato Grosso do Sul, Brazil

ABSTRACT

Leishmaniasis is a disease complex with various clinical symptoms caused by different species of parasites of the genus Leishmania. The visceral form of the disease, characterized by severe symptoms is fatal, if not treated. The high toxicity of current antileishmanial drugs and the need for long-term treatment make the therapy complicated, especially in a large number of infected children. Hence, the search for new therapies must be intensified. Oral administration of the trace element zinc has been considered in alternative treatments against different clinical forms of leishmaniasis. This study revealed that the administration of zinc in children with visceral leishmaniasis, during treatment with amphotericin B or glucantime, accelerates the regression of the spleen enlargement without interfering with the recovery of hematological parameters.

KEYWORDS: Zinc; Visceral leishmaniasis; Splenomegaly; Leishmaniasis treatment

INTRODUCTION

Visceral leishmaniasis (VL) is a zoonotic infectious disease which is caused in Americas by the protozoan parasite Leishmania infantum (L. infantum) transmitted through the bite of female infected phlebotomine sand flies Lutzomyia longipalpis (L. longipalpis)1. According to the World Health Organization (WHO), VL causes more than 20,000 deaths each year worldwide2. Among symptomatic patients, the main clinical signals of VL include intermittent fever, weight loss, adenopathy, anemia and substantial enlargement of the spleen and liver. In advanced stages of the disease, a consequence of the increase in the spleen and liver is intense abdominal distention and pain3. If patients are left untreated, the mortality rate can reach 100% within 2 years4.

Despite the high toxicity, the need for intravenous administration and the need for hospitalization and monitoring during treatment, the antileishmanial drugs amphotericin B and glucantime are used as first-line drugs in several countries5,6. Nowadays, many studies are conducted to determine alternative drugs to treat neglected diseases with lower toxicity and costs and to identify methods to increase the efficacy of current drugs, decreasing the side effects7,8. The efficacy of the trace element zinc (Zn) has been tested in a murine model of infection by L. major9 and in human cases of cutaneous leishmaniasis10. Since Zn plays an essential role in improving the nutritional and immunological status11, considered crucial parameters for the clinical progression of patients with VL in response to treatment, in this study we evaluated the role of zinc supplementation, combined with conventional antileishmanial therapy, in the regression of hepatomegaly and splenomegaly and recovery of hematological parameters in children with VL.

PATIENTS AND METHODS

Patients and Ethics Statement

Fifty-two patients with VL and fifteen healthy children aged 9–144 months were recruited to this study from the Hospital Maria Aparecida Pedrossian – HUMAP, Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brazil. VL was suspected in patients with fever and splenomegaly. Diagnosis was confirmed through detection of amastigote forms of the parasite in bone marrow aspiration samples. The healthy children included in this study were recruited during pediatric queries for routine examination. Patients with other diseases were excluded. Prior to the recruitment of patients, the study was approved by the Research Ethics Committee of the University (Protocol N° 2207/0305.0.049.000-11). Written informed consents were obtained from parents or legal guardians of the minors.

Treatment and zinc supplementation

All patients were treated with amphotericin B (0.5–1 mg/kg/day administered intravenously) or meglumine antimoniate (glucantime®) (20 mg/kg/day administered intravenously) for 20 days. During treatment, patients were divided into two groups: one group received conventional treatment alone, while the other group received both conventional treatment and a syrup containing zinc in the form of a chelate solution (10 mg/ml). The group received a total dose of 2 mg/kg/day in 14 days.

Plasma zinc analysis

On days 1, 7 and 14 of treatment, blood samples were collected in polypropylene syringes and immediately transferred to vacuum tubes free of trace elements (BD Vacutainer Systems). Plasma samples were separated by centrifugation and stored at −20 °C for analytical analysis. All plastic or glass materials were previously treated for decontamination. The calibration curves were constructed using four different zinc concentrations with values ranging from 0.25-2.0 mg/L. Atomic absorption analyses for plasma zinc were performed using a Perkin Elmer AA100 spectrometer (Waltham, MA, USA).

Liver and spleen measurement

On days 1, 7 and 14 of treatment, liver and the spleen enlargement were measured using percussion methods and a flexible measuring tape. Measurements for all patients were performed by the same researcher during the entire study period. The liver was measured at the level of the right hemiclavicular line, and the spleen was measured at the level of the transverse line drawn perpendicular to the left costal margin. The longitudinal and transversal lengths of the spleen were used to calculate the area of the organ using the formula of an elliptic area.

Classification of the nutritional status

The nutritional status was classified based on the World Health Organization (WHO) weight-for-age growth charts12 used for monitoring the growth of children.

Serum albumin quantification

On days 1, 7 and 14 after the beginning of treatment, blood samples were collected in collection tubes (BD microtainer®), serum samples were separated by centrifugation and the albumin levels were measured using a specific kit (ALB2 Roche®) according to the manufacturer's instructions.

Total blood count

On days 1, 7 and 14 after the beginning of treatment, blood samples were collected intravenously from all patients using specific blood collection tubes (BD microtainer®). Hematologic parameters were assessed in a Sysmex XN3000™ hematology analyzer.

Statistical analysis

Data were expressed as means ± SEM. The statistical significances between different time points were calculated using the Student's t test. All analyses were performed using the PRISM 5.0 software. Results were considered statistically different when the p value was < 0.05.

RESULTS AND DISCUSSION

Before the beginning of treatment, plasma Zn levels in all patients was measured (Figure 1A). Some studies showed that plasma Zn levels in patients with VL were lower than those of healthy individuals13,14. Our results have shown that plasma Zn levels were lower in patients supplemented with Zn than in patients who did not receive Zn supplementation, despite this, none of the groups presented plasmatic Zn levels lower than healthy children (Figure 1A). Even when Zn levels are within the normal range, patients can receive the mineral supplementation as a preventive measure15. Moreover, less than 0.2% of the total body Zn content circulates are present in plasma samples16; hence, it may not be a reliable indicator to assess the Zn levels in tissues. The supplementation of Zn was performed in a blind manner, and the quantification of the trace element on days 1, 7 and 14 after the beginning of supplementation showed that plasma Zn levels gradually increased (Figure 1B), while it remained unchanged in patients who did not receive Zn supplementation (Figure 1C).

Figure 1 A) Initial plasma zinc levels quantificated in healthy children (n=15) and in patients with visceral leishmaniasis before the beginning of zinc supplementation (n=23) or in those who did not receive zinc supplementation (n=29); B) Plasmatic zinc levels quantificated on days 1, 7, and 14 of zinc supplementation and treatment with amphotericin B or glucantime in patients with visceral leishmaniasis (n=23); C) Plasmatic zinc levels quantificated on days 1, 7, and 14 of treatment with amphotericin B or glucantime in patients with visceral leishmaniasis who did not receive zinc supplementation (n=29); D) Serum albumin levels of healthy children and patients with visceral leishmaniasis with and without zinc supplementation on days 1, 7, and 14 of treatment with amphotericin B or glucantime. The reference interval was delimited by dashed lines according to the values proposed by the kit manufacturer. *p< 0.05 in A, B and D, #p< 0.05 compared with healthy controls in D and ns=not statistically different in A–C 

The mean age and gender were not significantly different between children who received Zn supplementation and those who did not receive Zn supplementation during treatment of VL (Table 1). Previous studies showed that VL in Brazil has a great incidence in children17,18. Although literature suggests predominance of the disease in male gender19, our data are in accordance with the results of Queiroz et al.'s study20, who found the same distribution of the disease between children of both genders. An important risk factor of severe VL is malnutrition21. In this study, most of the recruited patients had normal nutritional status based on the weight-for-age indicator in the WHO growth charts (Table 1). Although serum albumin levels were used to evaluate the nutritional status, albumin levels are not specific to detect malnutrition and can be affected by some factors, such as inflammation22. Similarly to the results obtained by Gatto et al.23, here we showed that serum albumin is slightly reduced during VL and the treatment of children, whether using amphotericin B or glucantime alone or in combination with Zn supplementation, increases serum albumin to normal levels (Figure 1D).

Table 1 Age, gender and nutritional status of patients with VL and healthy children enrolled in the study 

With Zn Without Zn Healthy
Mean age (months) 46.20 ± 9.66 43.76 ± 6.50 44.60 ± 10.20
Male n=11 n=11 n=06
Female n=12 n=18 n=09
Nutritional status Number/Total (%)
Overweight 1/23 (4.35) 1/29 (3.45) 0
Overweight risk 2/23 (8.69) 3/29 (10.34) 0
Normal nutrition 18/23 (78.26) 24/29 (82.76) 15/15 (100)
Nutritional risk 1/23 (4.35) 1/29 (3.45) 0
Malnutrition 1/23 (4.35) 0 0

The earliest signs of improvement in the condition of patients with VL are the regression of clinical symptoms and splenomegaly. This study revealed that patients who received Zn supplementation during treatment with amphotericin B or glucantime showed rapid reduction in the spleen size compared with those patients who did not receive Zn (Figure 2A). On day 7 of treatment, patients supplemented with Zn had a significant reduction in the area of spleen, while patients without Zn supplementation presented considerable regression of splenomegaly only 20 days after the beginning of treatment (Figure 2A). The reduction of hepatomegaly was also evaluated in patients. On day 7 of treatment, patients supplemented with Zn presented a slight reduction in the liver size (Figure 2B); however, a significant reduction occurred in both groups of patients on day 14 of treatment (Figure 2B). In our model, we found that Zn supplementation only accelerates the splenomegaly regression but not the hepatomegaly in patients with VL. However, we believe that these data are still interesting as most studies showed that splenomegaly is more commonly observed than hepatomegaly among patients with VL. Enlargement of spleen is reported in about 90% of patients24, while the liver involvement is observed in less than 30% of patients25.

Figure 2 A) Area of spleen; B) size of liver; C) number of leukocytes; D) hemoglobin levels evaluated in patients with visceral leishmaniasis with (n=23) or without zinc supplementation (n=29) during treatment with amphotericin B or glucantime on days 1, 7, and 14 of treatment. *p< 0.05 and ns=not statistically different 

During the treatment of VL, recovery of hematological parameters were among the signs that indicated an improvement in patients' conditions26; hence, in this study, leukocytes and hemoglobin levels were evaluated in blood samples of patients. Treatment with amphotericin B or glucantime increased leukocyte and hemoglobin levels in both groups (Figures 2C and 2D). Moreover, Zn supplementation did not interfere with hematological parameters of patients during treatment of VL (Figures 2C and 2D). Our data are not in agreement with those of Dunchateau et al.27, who showed that Zn supplementation increased the circulating number of T lymphocytes in elderly people. This disagreement can be explained by the high concentration of Zn administrated by Dunchateau et al.27 (400 mg/day) and because elderly patients were used in that study. On contrary, the results of our study are in accordance with those of Bonham et al.28 who found that supplementation with 30 mg/kg/day of Zn in adult healthy men had no effect on the circulating levels of peripheral blood leukocytes subsets.

Previous and recent studies have shown that, although both antileishmanial drugs are effective, children treated with amphotericin B showed faster clinical improvement and shorter hospital stay than those children treated with glucantime29,30. The main advantage of glucantime compared with amphotericin B is the lower cost, for this reason most patients in our study were treated with glucantime. Only a few patients received amphotericin B treatment, thus, we could not evaluate whether the choice of drug interfered with the results. Future studies are necessary to evaluate whether Zn supplementation has different results when used with amphotericin B or with glucantime alone.

Our data corroborate the importance of zinc supplementation favoring the regression of splenomegaly in children during treatment of VL. These findings open new perspectives to increase the effectiveness of the current treatment of VL.

FUNDING

This study received funding from the National Council for Scientific and Technological Development (CNPq) (Process N° 304946/2013-3) and from the Foundation for the Support and Development of Education, Science and Technology of the State of Mato Grosso do Sul (FUNDECT) (Process N° 0179/12).

ACKNOWLEDGMENTS

We are grateful to the staffs of the Clinical Analysis Laboratory and of the Pediatric and Intensive Care Service of the Maria Aparecida Pedrossian Hospital. We also thank Paula Cristhina Niz Xavier for her technical assistance.

REFERENCES

1. Maurício IL, Stothard JR, Miles MA. The strange case of Leishmania chagasi. Parasitol Today. 2000;16:188-9 [ Links ]

2. World Health Organization. Leishmaniasis. [cited 2018 Aug 8]. Available from: http://www.who.int/leishmaniasis/en/Links ]

3. Herwaldt BL. Leishmaniasis. Lancet. 1999;354:1191-9. [ Links ]

4. World Health Organization. Leishmaniasis: clinical forms of the leishmaniases. [cited 2018 Aug 8]. Available from: http://www.who.int/leishmaniasis/disease/clinical_forms_leishmaniases/en/index2.htmlLinks ]

5. Mishra M, Biswas UK, Jha AM, Khan AB. Amphotericin versus sodium stibogluconate in first-line treatment of Indian kalaazar. Lancet. 1994;344:1599-600. [ Links ]

6. Sundar S, Singh A. Recent developments and future prospects in the treatment of visceral leishmaniasis. Ther Adv Infect Dis. 2016;3:98-109. [ Links ]

7. Mendonça DV, Martins VT, Lage DP, Dias DS, Ribeiro PA, Carvalho AM, et al. Comparing the therapeutic efficacy of different amphotericin B-carrying delivery systems against visceral leishmaniasis. Exp Parasitol. 2018;186:24-35. [ Links ]

8. Hendrickx S, Van den Kerkhof M, Mabille D, Cos P, Delputte P, Maes L, et al. Combined treatment of miltefosine and paromomycin delays the onset of experimental drug resistance in Leishmania infantum. PLoS Negl Trop Dis. 2017;11:e0005620. [ Links ]

9. Afshari M, Riazi-Rad F, Khaze V, Bahrami F, Ajdary S, Alimohammadian MH. Oral treatment with zinc sulfate increases the expression of Th1 cytokines mRNA in BALB/c mice infected with Leishmania major. Cytokine. 2016;81:71-6. [ Links ]

10. Farajzadeh S, Hakimi Parizi M, Haghdoost AA, Mohebbi A, Mohammadi S, Pardakhty A, et al. Comparison between intralesional injection of zinc sulfate 2 % solution and intralesional meglumine antimoniate in the treatment of acute old world dry type cutaneous leishmaniasis: a randomized double-blind clinical trial. J Parasit Dis. 2016;40:935-9. [ Links ]

11. Prasad AS. Discovery of human zinc deficiency: its impact on human health and disease. Adv Nutr. 2013;4:176-90. [ Links ]

12. World Health Organization. Child growth standards. [cited 2018 Aug 8]. Available from: http://www.who.int/childgrowth/standards/weight_for_age/en/Links ]

13. Van Weyenbergh J, Santana G, D'Oliveira Júnior A, Santos Júnior AF, Costa CH, Carvalho EM, et al. Zinc/copper imbalance reflects immune dysfunction in human leishmaniasis: an ex vivo and in vitro study. BMC Infect Dis. 2004;4:50 [ Links ]

14. Mishra J, Carpenter S, Singh S. Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India. Indian J Med Res. 2010;131:793-8 [ Links ]

15. Yakoob MY, Theodoratou E, Jabeen A, Imdad A, Eisele TP, Ferguson J, et al. Preventive zinc supplementation in developing countries: impact on mortality and morbidity due to diarrhea, pneumonia and malaria. BMC Public Health. 2011;11 Suppl 3:S23. [ Links ]

16. Pinna K, Woodhouse LR, Sutherland B, Shames DM, King JC. Exchangeable zinc pool masses and turnover are maintained in healthy men with low zinc intakes. J Nutr. 2001;131:2288-94. [ Links ]

17. Campos Júnior D. Características clínico-epidemiológicas do Calazar na criança: estudo de 75 casos. J Pediatr (Rio J). 1995;71:261-5. [ Links ]

18. Costa CH, Pereira HF, Araújo MV. Epidemia de leishmaniose visceral no Estado do Piauí, Brasil, 1980-1986. Rev Saude Publica. 1990;24:361-72. [ Links ]

19. Pastorino AC, Jacob CM, Oselka GW, Carneiro-Sampaio MM. Visceral leishmaniasis: clinical and laboratorial aspects. J Pediatr (Rio J). 2002;78:120-7. [ Links ]

20. Queiroz MJ, Alves JG, Correia JB. Visceral leishmaniasis: clinical and epidemiological features of children in an endemic area. J Pediatr (Rio J). 2004;80:141-6. [ Links ]

21. Carrillo E, Jimenez MA, Sanchez C, Cunha J, Martins CM, da Paixão Sevá A, et al. Protein malnutrition impairs the immune response and influences the severity of infection in a hamster model of chronic visceral leishmaniasis. PLoS One. 2014;9:e89412 [ Links ]

22. Ishida S, Hashimoto I, Seike T, Abe Y, Nakaya Y, Nakanishi H. Serum albumin levels correlate with inflammation rather than nutrition supply in burns patients: a retrospective study. J Med Invest. 2014;61:361-8 [ Links ]

23. Gatto M, de Abreu MM, Tasca KI, Simão JC, Fortaleza CM, Pereira PC, et al. Biochemical and nutritional evaluation of patients with visceral leishmaniasis before and after treatment with leishmanicidal drugs. Rev Soc Bras Med Trop. 2013;46:735-40 [ Links ]

24. Rocha NA, Silva GB, Oliveira MJ, Abreu KL, Franco LF, Silva MP, et al. Visceral leishmaniasis in children: a cohort of 120 patients in a metropolitan city of Brazil. Turk J Pediatr. 2011;53:154-60. [ Links ]

25. Chouchene S, Braham N, Bouatay A, Hizem S, Berriri S, Eljemai A, et al. Anomalies hématologiques au cours de la leishmaniose viscérale infantile. Arch Pediatr. 2015;22:1107-11. [ Links ]

26. Varma N, Naseem S. Hematologic changes in visceral leishmaniasis/kala azar. Indian J Hematol Blood Transfus. 2010;26:78-82. [ Links ]

27. Duchateau J, Delepesse G, Vrijens R, Collet H. Beneficial effects of oral zinc supplementation on the immune response of old people. Am J Med. 1981;70:1001-4. [ Links ]

28. Bonham M, O'Connor JM, Alexander HD, Coulter J, Walsh PM, McAnena LB, et al. Zinc supplementation has no effect on circulating levels of peripheral blood leucocytes and lymphocyte subsets in healthy adult men. Br J Nutr. 2003;89:695-703. [ Links ]

29. Kafetzis DA, Velissariou IM, Stabouli S, Mavrikou M, Delis D, Liapi G. Treatment of paediatric visceral leishmaniasis: amphotericin B or pentavalent antimony compounds? Int J Antimicrob Agents. 2005;25:26-30. [ Links ]

30. Apa H, Devrim, Bayram N, Deveci R, Demir-Özek G, Carti ÖU. Lipossomal amphotericin B versus pentavalent antimony salts for visceral Leishmania in children. Turk J Pediatr. 2013;55:378-83. [ Links ]

Recibido: 22 de Junio de 2018; Aprobado: 09 de Agosto de 2018

Correspondence to: Maria Cláudia Silva Universidade Católica Dom Bosco, Programa de Pós-Graduação em Biotecnologia, Avenida Tamandaré, 6000, CEP 79117-900, Campo Grande, MS, Brazil Tel: +55 67 3312-3448E-mail:mariaclaudiasilva@usp.br

CONFLICT OF INTERESTS

The authors declare that the research was conducted without conflicts of interest.

AUTHORS' CONTRIBUTIONS

Conceived and designed the experiments: DCBC, LZGZ, MCS; patient's recruitment and collection of human samples: DCBC, LZGZ; writing – original draft: MCS, DCBC; writing – review and editing: DCBC, LZGZ, FZC, SCS, VQR, KTCM, CMEC, MCS; analyzed the data: MCS, DCBC; statistics: MCS.

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.