439 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 5, 1977.
Granulocyte
function
in visceral leishmaniasis
G. SCHILIRO, A. Russo, L. MAURO, S. MUSUMECI AND G. Russo Departments of Paediatrics and Hygiene, University of Catania, Catania, Italy
Summary Nitroblue tetrazolium test in nine children with visceral leishmaniasis (VL) showed no increase of spontaneous reduction dy nkutrophils. The normal bactericidal activity of four of these patients excludes an impaired function of the neutrophils in VL. Introduction Visceral leishmaniasis still occurs as endemic disease in Sicily with the usual characteristics of anaemia, leucopenia and thrombocytopenia (Russo et al., 1975). The granulocyte count is decreased. It is still controversial if neutropenia is due to increased marginal pool, splenic sequestration, reduced granulocyte half life or to a combination of these mechanisms (MUSUMECI et al., 1977). No studies are available about the function of granulocytes in visceral leishmaniasis. Although several authors have described an increased spontaneous reduction of nitroblue-tetrazolium (NBT) by neutrophils in malaria and other parasitic diseases (MATULA & PATERSON, 1971; ANDERSEN, 1971; CHRETIEN & GARAGUSI, 1971; PUJOL-MOIX, 1971), this aspect of
granulocyte function has not yet been studied in visceral leishmaniasis. In this paper we report the results of a study on the bactericidal activity and the NBT test in this disease. Patients and methods Nine children from 11 months to six years of age were studied. In all cases the disease had the tvnical uicture of irregular fever and spleen enlarger&& ^anaemia, thrombocytopenia and neutropenia. In all cases the diagnosis was confirmed by finding leishmania in the bone marrow. The NBT test was performed as follows: 10 ml of heparinized blood (100 U/ml blood) collected in a plastic syringe, were centrifuged in a polypropylene test tube and 0.1 ml of the leucocyte layer was mixed with 0.1 ml of a 0.1% solution of NBT in physiological saline. This mixture was gently shaken and incubated at 37°C for 15 minutes and then kept at room temperature for an additional 15 minutes. Slides of the incubated material were prepared and stained with Giemsa solution. The slides were evaluated under oil immersion and 100 neutrophils were examined. The score was expressed as the number of cells containing blue-black formazan granules per 100 neutrophils. In four of our patients the bactericidal activity of the neutrophils for Staphylococcus aureus A.T.C.C. 6538 was investigated by the method of QUIE et al. (1967). Because of neutropenia, 15 to 20 ml of blood were necessary in order to obtain a sufficient concentration. Every patient was tested together with a control subject hospitalized for non-infectious conditions and
another with untreated bacterial infection. The children in the former control group were suspected to have thalassaemia or other haemoglobinopathies. Results All the children with visceral leishmaniasis showed NBT scores within the range of the normal controls (2 to 13%). All the subjects with untreated bacterial infections presented, as expected, an increased score. The bacterial activity of the neutrophils was normal in the four patients in whom it was studied. Complete results of NBT test and bactericidal activity are reported in the Table. Table-Results of NBT test and bactericidal activity in visceral leishmaniasis Subjects 1 2 3 4 5 6 7 8 9 Normal controls (mean) Acute bacterial infections (mean)
NBT score Bactericidal activity* % 6 2 8 6 6 90-95 8 90-95 9 90-95 4 90-95 5 9.1*4.0 90-95 341-8.1
*Percentage of Staphylococci killed at 60 minutes. Discussion The increased spontaneous reduction of NBT by peripheral blood neutrophils was first described by PARK et al. (1968) in bacterial infections. However, increased NBT scores have also been reported in non-bacterial diseases such as mycoplasma and fungal infections (FREEMAN & KING, 1972), malaria and other parasitic diseases (MATULA & PATERSON, 1971; ANDERSEN, 1971; CHRETIEN & GARAGUSI, 1971; PUJOL-MOIX, 1971). In view
of these data, NBT score was expected to be increased in visceral leishmaniasis. Our results show, however, scores within the normal range. Because this could be a “false-negative” result, secondary to defective neutrophi1 function (PARK & GOOD, 1970), we studied the bactericidal activity of these cells in four of our subjects. The normal bactericidal function is in agreement with earlier studies on the mechanism of neutropenia (MUSUMECI et al., 1977) which does not seem related to
440
GRANULOCYTE
FUNCTION
any depression of metabolism of granulocytes, and suggests that the normal NBT scores are valid. The result questions the interpretation of the raised NBT scores in malaria which, in some ways, is very similar to visceral leishmaniasis. It is possible that factors which stimulate the neutrophil activity in malaria are absent in visceral leishmaniasis. References Andersen, B. R. (1971). NBT test in malaria. Lancet, ii, 317. Chretien, J. H. & Garagusi, V. F. (1971). NBT test in parasitic disease. Lancet, ii, 549. Freeman, R. & Ring, B. (1972). NBT test and mycoplasma. Lancet, i, 962. Matula, G. & Paterson, P. Y. (1971). Spontaneous in vitro reduction of nitroblue tetrazolium by neutrophils of adult patients with bacterial infection. New England Journal of Medicine, 285,3 1l-3 16. Musumeci, S., D’Agata, A., Schilirb, G. & Fischer, A.
IN VISCERAL
LEISHMANIASIS
(1977). Studies of the neutropenia in kala-azar: results in two patients. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70,500-503.
Park, B. H., Fikrig, S. M. & Smithwick, E. M. (1968). Infection and nitroblue-tetrazolium reduction by neutrophils. Lancet, ii, 532-534. Park, B. H. & Good, R. A. (1970). NBT test stimulated. Lance& ii, 616. Pujol-Moix, M. N. (1971). NBT test in malaria. Lancet, ii, 871-872.
Quie, P. G., White, J. G., Holmes, B. & Good, R. A. (1967). In vitro bactericidal capacity of human polymorphonuclear leucocytes: diminished activity in chronic granulomatous disease of childhood. Journal of Clinical Znvestigation, 46, 668-672.
Russo, G., Musumeci, S., Russo, A. & Schilirb, G. (1975). Visceral leishmaniasis in Italy. Lance& i, 640. Accepted for publication
28th May, 1977.
Granulocyte
function
in visceral leishmaniasis
G. SCHILIRO, A. Russo, L. MAURO, S. MUSUMECI AND G. Russo Departments of Paediatrics and Hygiene, University of Catania, Catania, Italy
Summary Nitroblue tetrazolium test in nine children with visceral leishmaniasis (VL) showed no increase of spontaneous reduction dy nkutrophils. The normal bactericidal activity of four of these patients excludes an impaired function of the neutrophils in VL. Introduction Visceral leishmaniasis still occurs as endemic disease in Sicily with the usual characteristics of anaemia, leucopenia and thrombocytopenia (Russo et al., 1975). The granulocyte count is decreased. It is still controversial if neutropenia is due to increased marginal pool, splenic sequestration, reduced granulocyte half life or to a combination of these mechanisms (MUSUMECI et al., 1977). No studies are available about the function of granulocytes in visceral leishmaniasis. Although several authors have described an increased spontaneous reduction of nitroblue-tetrazolium (NBT) by neutrophils in malaria and other parasitic diseases (MATULA & PATERSON, 1971; ANDERSEN, 1971; CHRETIEN & GARAGUSI, 1971; PUJOL-MOIX, 1971), this aspect of
granulocyte function has not yet been studied in visceral leishmaniasis. In this paper we report the results of a study on the bactericidal activity and the NBT test in this disease. Patients and methods Nine children from 11 months to six years of age were studied. In all cases the disease had the tvnical uicture of irregular fever and spleen enlarger&& ^anaemia, thrombocytopenia and neutropenia. In all cases the diagnosis was confirmed by finding leishmania in the bone marrow. The NBT test was performed as follows: 10 ml of heparinized blood (100 U/ml blood) collected in a plastic syringe, were centrifuged in a polypropylene test tube and 0.1 ml of the leucocyte layer was mixed with 0.1 ml of a 0.1% solution of NBT in physiological saline. This mixture was gently shaken and incubated at 37°C for 15 minutes and then kept at room temperature for an additional 15 minutes. Slides of the incubated material were prepared and stained with Giemsa solution. The slides were evaluated under oil immersion and 100 neutrophils were examined. The score was expressed as the number of cells containing blue-black formazan granules per 100 neutrophils. In four of our patients the bactericidal activity of the neutrophils for Staphylococcus aureus A.T.C.C. 6538 was investigated by the method of QUIE et al. (1967). Because of neutropenia, 15 to 20 ml of blood were necessary in order to obtain a sufficient concentration. Every patient was tested together with a control subject hospitalized for non-infectious conditions and
another with untreated bacterial infection. The children in the former control group were suspected to have thalassaemia or other haemoglobinopathies. Results All the children with visceral leishmaniasis showed NBT scores within the range of the normal controls (2 to 13%). All the subjects with untreated bacterial infections presented, as expected, an increased score. The bacterial activity of the neutrophils was normal in the four patients in whom it was studied. Complete results of NBT test and bactericidal activity are reported in the Table. Table-Results of NBT test and bactericidal activity in visceral leishmaniasis Subjects 1 2 3 4 5 6 7 8 9 Normal controls (mean) Acute bacterial infections (mean)
NBT score Bactericidal activity* % 6 2 8 6 6 90-95 8 90-95 9 90-95 4 90-95 5 9.1*4.0 90-95 341-8.1
*Percentage of Staphylococci killed at 60 minutes. Discussion The increased spontaneous reduction of NBT by peripheral blood neutrophils was first described by PARK et al. (1968) in bacterial infections. However, increased NBT scores have also been reported in non-bacterial diseases such as mycoplasma and fungal infections (FREEMAN & KING, 1972), malaria and other parasitic diseases (MATULA & PATERSON, 1971; ANDERSEN, 1971; CHRETIEN & GARAGUSI, 1971; PUJOL-MOIX, 1971). In view
of these data, NBT score was expected to be increased in visceral leishmaniasis. Our results show, however, scores within the normal range. Because this could be a “false-negative” result, secondary to defective neutrophi1 function (PARK & GOOD, 1970), we studied the bactericidal activity of these cells in four of our subjects. The normal bactericidal function is in agreement with earlier studies on the mechanism of neutropenia (MUSUMECI et al., 1977) which does not seem related to
440
GRANULOCYTE
FUNCTION
any depression of metabolism of granulocytes, and suggests that the normal NBT scores are valid. The result questions the interpretation of the raised NBT scores in malaria which, in some ways, is very similar to visceral leishmaniasis. It is possible that factors which stimulate the neutrophil activity in malaria are absent in visceral leishmaniasis. References Andersen, B. R. (1971). NBT test in malaria. Lancet, ii, 317. Chretien, J. H. & Garagusi, V. F. (1971). NBT test in parasitic disease. Lancet, ii, 549. Freeman, R. & Ring, B. (1972). NBT test and mycoplasma. Lancet, i, 962. Matula, G. & Paterson, P. Y. (1971). Spontaneous in vitro reduction of nitroblue tetrazolium by neutrophils of adult patients with bacterial infection. New England Journal of Medicine, 285,3 1l-3 16. Musumeci, S., D’Agata, A., Schilirb, G. & Fischer, A.
IN VISCERAL
LEISHMANIASIS
(1977). Studies of the neutropenia in kala-azar: results in two patients. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70,500-503.
Park, B. H., Fikrig, S. M. & Smithwick, E. M. (1968). Infection and nitroblue-tetrazolium reduction by neutrophils. Lancet, ii, 532-534. Park, B. H. & Good, R. A. (1970). NBT test stimulated. Lance& ii, 616. Pujol-Moix, M. N. (1971). NBT test in malaria. Lancet, ii, 871-872.
Quie, P. G., White, J. G., Holmes, B. & Good, R. A. (1967). In vitro bactericidal capacity of human polymorphonuclear leucocytes: diminished activity in chronic granulomatous disease of childhood. Journal of Clinical Znvestigation, 46, 668-672.
Russo, G., Musumeci, S., Russo, A. & Schilirb, G. (1975). Visceral leishmaniasis in Italy. Lance& i, 640. Accepted for publication
28th May, 1977.
