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Rapid clearance of circulating Leishmania kinetoplast DNA after treatment of visceral leishmaniasis
Acta Tropica 92 (2004) 279–283
Short communication
Rapid clearance of circulating Leishmania kinetoplast DNA after treatment of visceral leishmaniasis Jolande Discha , Maria Cl´audia Oliveiraa , Marcela Orsinia,b , Ana Rabelloa,∗ a
b
Laborat´orio de Pesquisas Cl´ınicas, Centro de Pesquisas Ren´e Rachou-FIOCRUZ. Av. Augusto de Lima, 1715, 30190-002, Belo Horizonte, MG, Brazil. Departamento de Cl´ınica M´edica, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Received 12 April 2004; received in revised form 3 August 2004; accepted 9 August 2004 Available online 11 September 2004
Abstract With the aim of evaluating the utility of the detection of Leishmania kDNA in peripheral blood for the cure assessment of visceral leishmaniasis (VL), a PCR based method was performed in patients with confirmed VL at three follow-up periods after specific chemotherapy with pentavalent antimonial. In 16 out of 17 (94.1%) patients with pre-treatment detectable kDNA that were clinically cured, the PCR turned negative up to 37 days after the initiation of treatment, remaining negative over 90 days after treatment. The clearance of Leishmania kDNA from peripheral blood of patients with VL hints to occur during or shortly after treatment concurring or preceding clinical recovery. © 2004 Elsevier B.V. All rights reserved. Keywords: Visceral leishmaniasis; Diagnosis; PCR; Treatment
1. Introduction Visceral leishmaniasis (VL) is a systemic disease caused by protozoa parasites pertaining to the Leishmania donovani complex: Leishmania (Leishmania) donovani, L (L.) chagasi and L. (L.) infantum. It is endemic in 62 countries, with a total of 200 million people at risk, estimated 500,000 new cases each year world∗ Corresponding author. Tel.: +55 31 3295 3566x108; fax: +55 31 3295 3115. E-mail address: [email protected] (A. Rabello).
0001-706X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.08.002
wide (Desjeux, 1996) and 41,000 recorded deaths in the year 2000 (WHO, 2001). The main areas of concern are Sudan, Eastern India, Brazil and the Mediterranean zone. In the vertebrate host, Leishmania reproduces in cells of the phagocytic mononuclear system as intracellular amastigotes, involves organs such as spleen, liver and bone marrow and leads to a febrile hepatosplenic syndrome accompanied by a decrease in the number of erythrocytes, leukocytes and platelets. The disease may progress towards severe complications, usually bacterial infections and bleeding, and is fatal if untreated. Definition of clinical cure is mostly based on clinical
280
J. Disch et al. / Acta Tropica 92 (2004) 279–283
improvement denoted by the vanishing of fever, reduction of spleen and liver sizes and normalization of blood cell counts. The parasitological tools are invasive and therefore not practical for clinical monitoring of therapeutic response and antibodies may persist for years after cure. Positive intradermal skin test is a sign of cellular adequate immune response that usually develops months after clinical recovery. The detection of Leishmania DNA via polymerase chain reaction (PCR) is a definitive breakthrough for the diagnosis of VL. The Leishmania PCR assays using peripheral blood as clinical specimen showed to be a highly efficient non-invasive alternative with sensitivities varying from 82 to 100% (Smyth et al., 1992; Lachaud et al., 2000; Fisa et al., 2002; Disch et al., 2003). The value of the PCR for cure assessment of VL has been mostly evaluated in HIV co-infected patients (Piarroux et al., 1994; Costa et al., 1996; Lachaud et al., 2000; Cruz et al., 2002; Pizzuto et al., 2001; Fisa et al., 2002). Nuzum et al. (1995) and Cascio et al. (2002) have evaluated the PCR as a tool for cure assessment of immunocompetent patients from India and Italy, suggesting a correspondence between clinical improvement and clearance of Leishmania DNA from peripheral blood. In this work, a group of patients receiving specific treatment of VL was followed-up and the clearance of Leishmania kDNA in peripheral blood evaluated during and after treatment.
2. Patients and methods 2.1. Study-design and patients The study was conducted at the Reference Center for Leishmaniasis of the “Centro de Pesquisas Ren´e Rachou” – FIOCRUZ, Belo Horizonte, Brazil. Diagnosis of VL was defined when, besides clinical and epidemiological features, amastigotes were seen at giemsa stained smears or promastigote forms were isolated from culture of bone marrow aspirates. In case of negative parasitology but positive immunofluorescent antibody test (IFAT), diagnosis of VL was assured by clinical response following specific chemotherapy. All patients were treated with meglumine antimoniate (81 mg/ml of Sbv –Aventis Pharma Ltda, S˜ao Paulo, Brazil) by intravenous administration of
20 mg Sbv /kg/day during 30 days, according to the Brazilian Ministry of Health’s recommendations. To evaluate the clearance of Leishmania kDNA during and after treatment peripheral blood samples of 17 patients were grouped in three time-intervals: (i) the first one comprised the first 37 days after treatment initiation, encompassing the 30 days of treatment and the first 7 days after its termination; (ii) in the second period, clinical evaluation and blood samples were collected between 50 and 90 days after treatment initiation and; (iii) the third period included samples collected after 90 days after treatment initiation. During the follow-up occasions patients were re-examined to determine clinical recovery. The definition of clinical cure was based on absence of fever and the return of spleen, liver and blood cells to normal. The FIOCRUZ Ethical Review Board approved this research project. All patients signed the informed consent form. 2.2. DNA extraction For DNA extraction from peripheral blood cells the GenomicPrepTM Blood DNA Isolation Kit (Amersham Pharmacia Biotech, Inc. Piscataway, NJ, USA) was used according to manufactor’s instructions. In short, from 300 L of whole blood, red blood cells were lysed in a red cell lysis solution, followed by white blood cell lysis and their nuclei in nucleic lysis solution. Cellular proteins were removed by a salt precipitation step, which precipitates the proteins but leaves the DNA in solution. Finally, the DNA was concentrated and desalted by isopropanol precipitation. The final product was resuspended in rehydration solution. Extractions were performed in sets of maximal 10 samples, including one negative sample from a non-exposed voluntary and one positive sample from a patient with active VL. 2.3. PCR amplification. The entire conserved kDNA region (120 bp) was amplified using a primer pair described by Degrave et al. (1994). Briefly, reaction mixtures were prepared in 10 L volumes using 1 L of DNA sample in 10 mM Tris–HCl pH 8.6, 0.6 M of each primer, 0.75 units of Taq DNA polymerase (Promega, Madison, WI, USA), 50 mM KCl pH 8.3, 2 mM MgCl2 , and 200 M of dNTPs. Forty amplification cycles were undertaken
J. Disch et al. / Acta Tropica 92 (2004) 279–283
using a temperature of 95 ◦ C for denaturation, 63 ◦ C for annealing and 72 ◦ C for extension, 30 s each step. Each reaction set included two negative PCR controls (no DNA) and one positive control (30 fg of DNA extracted from L. (L.) chagasi promastigotes in culture, determined by UV 260 nm absorption). An isolated area, irradiated with UV light prior to reaction setup and free of DNA, biological samples or amplification products, was used to prepare the reactions. PCR was performed using a MJ Research thermo-cycler (PTC-100-60, Inc. Watertown, MA, USA). After amplification, 10 L of the sample were run in a 6% polyacrylamide gel in a Mini-Protean apparatus (Biorad, Hercules, CA) and stained with silver to visualize the products.
3. Results Table 1 lists the PCR results of 17 patients included in the study and depicts the individual results of the three follow-up intervals. All patients were clinically cured at different time-points after treatment and none of them relapsed during the follow-up period. In 16 out of 17 patients (94.1%) the PCR turned negative (Fig. 1) during the first follow-up evaluation
281
Fig. 1. PCR amplified products from the conserved region of Leishmania minicircle kDNA by polymerase chain reaction. NC: negative control (no DNA); lanes 1–3: blood samples from patrients with diagnosis of VL before treatment; lanes 4–10: blood samples from patients during the follow-up controls; lane 11: negative control (healthy individual); lane 12: positive PCR control (Leishmania DNA).
that comprised the period of 30 days treatment until 7 days after treatment. Clearance of kDNA occurred before or concomitant to clinical definition of cure, based on absence of fever, normal physical examination and normal blood cells count (bold figures in Table 1). Three patients (ESM-number 5; RAC-number 13 and TMC-number 17) out of eight from whom samples were obtained during the first 15 days of treatment were still positive PCR at 12, 14 and 14 days of treatment, respectively, but kDNA was undetectable in blood samples taken at days 36, 33 and 29 after
Table 1 Leishmania kDNA detected by PCR in peripheral blood seventeen patients with visceral leishmaniasis during and after specific chemotherapy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Patients
Sex
Age
During and until 7 days after treatment
50–90 days after treatment
>90 days after treatment
ACCS AAOD DO ESVS ESM EFAM GFG JAP JNSR MLK PCS RMM RAC SGS SAD TCAF TMC
F F M M M F F M M F F M M F M F F
27 36 41 22 22 14 21 43 42 45 14 08 34 01 15 06 04
N (26) P (30) N (14) P (23) N (37) N (30) P (12) N (36) N (33) N (7) N (33) N (30) N (37) N (7) N (35) N (28) N (29) N (9) N (30) P (14) N (33) N (11) N (25) N (35), N (33) P (14) N (29)
N (54) P (82) N (56) N (85) N (69) ND N (62) N (68) N (67) N (62) N (92) N (61) N (63) N (65) N (50) N (61) N (83)
N (162) P (112) P (145) N (226) N (142) N (219) ND ND N (153) N (166) ND ND N (183) N (97) N (183) N (109) N (106) ND N (114)
*Day 0 refers to the first day of treatment, P = positive PCR, N = negative PCR, ND = not done. Figures in parenthesis correspond to precise day of blood collection and figures in bold correspond to the day on which cure was clinically defined.
282
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treatment evaluation. Fluctuation of circulating DNA was observed in one patient (DO-number 3) that presented negative PCR at the 14th day of treatment, became positive again at day 23 but turned negative at day 37 and remained so up to 142 days of follow-up. PCR became negative in five out of eight patients from whom samples were available during the first 15 days of treatment. Further follow-up of these patients demonstrated that negative results were sustained. Only one patient (AAOD-number 2) scored positive by PCR during the first period of follow-up (30 days). This patient, a 37-year-old woman had a severe VL with hemoglobin of 3.0 g/dL, 1200 leucocytes and 76,000 platelets/mm3 . She had refused blood transfusion due to religion constraints. At the end of the treatment with meglumine antimoniate, a significant clinical improvement was observed and the absence of symptoms and signs was determined at 82 days after treatment. However, Leishmania kDNA could still be amplified from the patient’s blood up to 145 days after treatment initiation. Only 8 months after treatment the PCR turned negative. At the same time, skin test became positive (reaction of 2.2 cm of diameter). In this group of patients, anti-L. chagasi IgG antibody detected by the immunofluorescent assay, repeated at the same follow-up occasions presented a tendency for decreasing titers over the period but remained positive.
4. Discussion In this study, we observed a rapid clearance of Leishmania kDNA in peripheral blood samples during or soon after treatment of VL. A specific test that accurately assures cure and/or predicts recurrence of VL is not yet available and fiercely sought-after. The efficiency of Leishmania DNA detection in peripheral blood of patients with VL may offer a non-invasive highly sensitive tool to help the assessment of cure, although a number of uncertainties are still to be addressed. For instance, is the early presence of DNA a direct evidence of viable pathogens or could this evidence be puzzled by the amplification of intact free nucleic acids? Two previous reports evaluated the presence of Leishmania DNA as a cure assessment tool in immunocompetent patients. Nuzum et al. (1995) evaluated 13
Indian patients in whom drug treatment had apparently been successful by clinical criteria and observed 14 and 0% of blood PCR positive patients at 1 and 6 months after treatment, respectively. Cascio et al. (2002) observed that Leishmania DNA was cleared from the peripheral blood within a median of 6 days after the start of lipossomal anfotericin and 14 days after the start of meglumine antimoniate in ten Italian children with VL. In this study, that included repeated follow-up of treated patients, 94% (16/17) of the patients with positive PCR at pre-treatment evaluation became negative during the 30-day treatment period or within the first week after treatment (37 days). Further followup of these patients showed that negative results were sustained. It is not clear to us why one patient had detectable Leishmania DNA until a long time after clinical cure was attained. This case differs from the observed by Cascio et al. (2002), that followed a patient that persisted with positive blood PCR after 3 months after treatment but relapsed 15 days latter. These observations suggest that the persistence of parasite DNA in blood (as well the persistence of parasites in bone marrow) seem not to be an unequivocal indicator of further relapse that is multi-factorial determined. As all of these patients were successfully treated and none of them relapsed, a broad and definitive conclusion about the utility of the blood PCR on the prediction of cure and relapse was not possible. Only the accumulation of experience with this tool will provide this information. This low frequency of relapses or treatment failure and the rapid clearance of parasite DNA in immunocompetents diverge from the evolution of patients co-infected with HIV, which present lower rates of cure, higher frequency of relapses and frequent persistence of circulating DNA. For instance, Fisa et al. (2002) using a nested PCR detected Leishmania infantum genomic DNA, 1 month after treatment in 7 of 10 patients, eight of them infected with HIV. All five that relapsed belonged to the HIV-infected group. Through this study-protocol, it was possible to conclude that the clearance of Leishmania kDNA from peripheral blood of patients with visceral leishmaniasis hints to occur during or shortly after treatment, concurring or preceding clinical recovery. Our data reinforce that blood PCR constitutes a useful non-invasive method for monitoring the cure of visceral leishmaniasis.
J. Disch et al. / Acta Tropica 92 (2004) 279–283
References Cascio, A., Calattini, S., Colomba, C., Scalamogna, C., Galazzi, M., Pizzuto, M., Gramiccia, M., Titone, L., Corbellino, M., Antinori, S., 2002. Polymerase chain reaction in the diagnosis and prognosis of Mediterranean visceral leishmaniasis in immunocompetent children. Pediatrics 109, E27. Costa, J.M., Durand, R., Deniau, M., Rivollet, D., Izri, M., Houin, R., Vidaud, M., Bretagne, S., 1996. PCR enzyme-linked immunosorbant assay for diagnosis of leishmaniasis in human imunodeficiency virus infected patients. J. Clin. Microbiol. 34, 1831–1833. Cruz, I., Canavate, C., Rubio, J.M., Morales, M.A., Chicharro, C., Laugna, F., Jimenez-Mejias, M., Sirera, G., Videla, S., Alvar, J., 2002. A nested polymerase chain reaction for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans. R. Soc. Trop. Med. Hyg. 96, 185–189. Degrave, W., Fernandes, O., Campbell, D., Bozza, M., Lopes, U.G., 1994. Use of molecular probes and PCR for detection and typing of Leishmania—a mini-review. Mem. Inst. Oswaldo Cruz 89, 463–469. Desjeux, P., 1996. Leishmaniasis–Public Health aspects and control. Clin. Dermatol. 14, 417–423. Disch, J., Maciel, M.C., Oliveira, M., Orsini, M., Rabello, A., 2003. Detection of circulating Leishmania chagasi DNA for the noninvasive diagnosis of human infection. Trans. R. Soc. Trop. Med. Hyg. 97, 1–5. Fisa, R., Riera, C., Ribeira, E., G´allego, M., Port´us, M., 2002. A nested polymerase chain reaction for diagnosis and follow-up
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of human visceral leishmaniasis. Trans. R. Soc. Med. Hyg. 96, 191–194. Lachaud, L., Dereure, J., Chabbert, E., Reyenes, J., Mauboussin, J.M., Oziol, E., Dedet, J.P., Bastien, P., 2000. Optimized PCR using patient blood samples for diagnosis and follow-up of visceral leishmaniasis, with special reference to AIDS patients. J. Clin. Microbiol. 38, 236–240. Nuzum, E., White, F., Thakur, C., Dietze, R., Wages, J., Grogl, M., Berman, J., 1995. Diagnosis of symptomatic visceral leishmaniasis by use of the polymerase chain reaction on patient blood. J. Infect. Dis. 171, 751–754. Piarroux, R., Gamberelli, F., Dumon, H., Fontes, M., Dunan, S., Mary, C., Toga, B., Quilici, M., 1994. Comparison of PCR with direct examination of bone marrow aspiration, myeloculture and serology for the diagnosis of visceral leishmaniasis in immunocompromised patients. J. Clin. Microbiol. 32, 745–749. Pizzuto, M., Piazza, M., Sense, D., Scalamogna, C., Calttini, S., Corsico, L., Persico, T., Adriani, B., Magni, C., Guaraldi, G., Gaiera, G., Ludovisi, A., Gramiccia, M., Galli, M., Moroni, M., Corbellino, M., Antinori, S., 2001. Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type 1. J. Clin. Microbiol. 39, 357–361. Smyth, A.J., Ghosh, A., Hassan, M.Q., de Bruijn, M.H.L., Adhya, S., Mallik, K.K., Barker, D.C., 1992. Rapid and sensitive detection of Leishmania kinetoplast DNA form spleen and blood samples of kala-azar patients. Parasitology 105, 183–192. World Health Organisation, 2001. The world health report 2001. World Health Organisation, Geneva.
Short communication
Rapid clearance of circulating Leishmania kinetoplast DNA after treatment of visceral leishmaniasis Jolande Discha , Maria Cl´audia Oliveiraa , Marcela Orsinia,b , Ana Rabelloa,∗ a
b
Laborat´orio de Pesquisas Cl´ınicas, Centro de Pesquisas Ren´e Rachou-FIOCRUZ. Av. Augusto de Lima, 1715, 30190-002, Belo Horizonte, MG, Brazil. Departamento de Cl´ınica M´edica, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Received 12 April 2004; received in revised form 3 August 2004; accepted 9 August 2004 Available online 11 September 2004
Abstract With the aim of evaluating the utility of the detection of Leishmania kDNA in peripheral blood for the cure assessment of visceral leishmaniasis (VL), a PCR based method was performed in patients with confirmed VL at three follow-up periods after specific chemotherapy with pentavalent antimonial. In 16 out of 17 (94.1%) patients with pre-treatment detectable kDNA that were clinically cured, the PCR turned negative up to 37 days after the initiation of treatment, remaining negative over 90 days after treatment. The clearance of Leishmania kDNA from peripheral blood of patients with VL hints to occur during or shortly after treatment concurring or preceding clinical recovery. © 2004 Elsevier B.V. All rights reserved. Keywords: Visceral leishmaniasis; Diagnosis; PCR; Treatment
1. Introduction Visceral leishmaniasis (VL) is a systemic disease caused by protozoa parasites pertaining to the Leishmania donovani complex: Leishmania (Leishmania) donovani, L (L.) chagasi and L. (L.) infantum. It is endemic in 62 countries, with a total of 200 million people at risk, estimated 500,000 new cases each year world∗ Corresponding author. Tel.: +55 31 3295 3566x108; fax: +55 31 3295 3115. E-mail address: [email protected] (A. Rabello).
0001-706X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.08.002
wide (Desjeux, 1996) and 41,000 recorded deaths in the year 2000 (WHO, 2001). The main areas of concern are Sudan, Eastern India, Brazil and the Mediterranean zone. In the vertebrate host, Leishmania reproduces in cells of the phagocytic mononuclear system as intracellular amastigotes, involves organs such as spleen, liver and bone marrow and leads to a febrile hepatosplenic syndrome accompanied by a decrease in the number of erythrocytes, leukocytes and platelets. The disease may progress towards severe complications, usually bacterial infections and bleeding, and is fatal if untreated. Definition of clinical cure is mostly based on clinical
280
J. Disch et al. / Acta Tropica 92 (2004) 279–283
improvement denoted by the vanishing of fever, reduction of spleen and liver sizes and normalization of blood cell counts. The parasitological tools are invasive and therefore not practical for clinical monitoring of therapeutic response and antibodies may persist for years after cure. Positive intradermal skin test is a sign of cellular adequate immune response that usually develops months after clinical recovery. The detection of Leishmania DNA via polymerase chain reaction (PCR) is a definitive breakthrough for the diagnosis of VL. The Leishmania PCR assays using peripheral blood as clinical specimen showed to be a highly efficient non-invasive alternative with sensitivities varying from 82 to 100% (Smyth et al., 1992; Lachaud et al., 2000; Fisa et al., 2002; Disch et al., 2003). The value of the PCR for cure assessment of VL has been mostly evaluated in HIV co-infected patients (Piarroux et al., 1994; Costa et al., 1996; Lachaud et al., 2000; Cruz et al., 2002; Pizzuto et al., 2001; Fisa et al., 2002). Nuzum et al. (1995) and Cascio et al. (2002) have evaluated the PCR as a tool for cure assessment of immunocompetent patients from India and Italy, suggesting a correspondence between clinical improvement and clearance of Leishmania DNA from peripheral blood. In this work, a group of patients receiving specific treatment of VL was followed-up and the clearance of Leishmania kDNA in peripheral blood evaluated during and after treatment.
2. Patients and methods 2.1. Study-design and patients The study was conducted at the Reference Center for Leishmaniasis of the “Centro de Pesquisas Ren´e Rachou” – FIOCRUZ, Belo Horizonte, Brazil. Diagnosis of VL was defined when, besides clinical and epidemiological features, amastigotes were seen at giemsa stained smears or promastigote forms were isolated from culture of bone marrow aspirates. In case of negative parasitology but positive immunofluorescent antibody test (IFAT), diagnosis of VL was assured by clinical response following specific chemotherapy. All patients were treated with meglumine antimoniate (81 mg/ml of Sbv –Aventis Pharma Ltda, S˜ao Paulo, Brazil) by intravenous administration of
20 mg Sbv /kg/day during 30 days, according to the Brazilian Ministry of Health’s recommendations. To evaluate the clearance of Leishmania kDNA during and after treatment peripheral blood samples of 17 patients were grouped in three time-intervals: (i) the first one comprised the first 37 days after treatment initiation, encompassing the 30 days of treatment and the first 7 days after its termination; (ii) in the second period, clinical evaluation and blood samples were collected between 50 and 90 days after treatment initiation and; (iii) the third period included samples collected after 90 days after treatment initiation. During the follow-up occasions patients were re-examined to determine clinical recovery. The definition of clinical cure was based on absence of fever and the return of spleen, liver and blood cells to normal. The FIOCRUZ Ethical Review Board approved this research project. All patients signed the informed consent form. 2.2. DNA extraction For DNA extraction from peripheral blood cells the GenomicPrepTM Blood DNA Isolation Kit (Amersham Pharmacia Biotech, Inc. Piscataway, NJ, USA) was used according to manufactor’s instructions. In short, from 300 L of whole blood, red blood cells were lysed in a red cell lysis solution, followed by white blood cell lysis and their nuclei in nucleic lysis solution. Cellular proteins were removed by a salt precipitation step, which precipitates the proteins but leaves the DNA in solution. Finally, the DNA was concentrated and desalted by isopropanol precipitation. The final product was resuspended in rehydration solution. Extractions were performed in sets of maximal 10 samples, including one negative sample from a non-exposed voluntary and one positive sample from a patient with active VL. 2.3. PCR amplification. The entire conserved kDNA region (120 bp) was amplified using a primer pair described by Degrave et al. (1994). Briefly, reaction mixtures were prepared in 10 L volumes using 1 L of DNA sample in 10 mM Tris–HCl pH 8.6, 0.6 M of each primer, 0.75 units of Taq DNA polymerase (Promega, Madison, WI, USA), 50 mM KCl pH 8.3, 2 mM MgCl2 , and 200 M of dNTPs. Forty amplification cycles were undertaken
J. Disch et al. / Acta Tropica 92 (2004) 279–283
using a temperature of 95 ◦ C for denaturation, 63 ◦ C for annealing and 72 ◦ C for extension, 30 s each step. Each reaction set included two negative PCR controls (no DNA) and one positive control (30 fg of DNA extracted from L. (L.) chagasi promastigotes in culture, determined by UV 260 nm absorption). An isolated area, irradiated with UV light prior to reaction setup and free of DNA, biological samples or amplification products, was used to prepare the reactions. PCR was performed using a MJ Research thermo-cycler (PTC-100-60, Inc. Watertown, MA, USA). After amplification, 10 L of the sample were run in a 6% polyacrylamide gel in a Mini-Protean apparatus (Biorad, Hercules, CA) and stained with silver to visualize the products.
3. Results Table 1 lists the PCR results of 17 patients included in the study and depicts the individual results of the three follow-up intervals. All patients were clinically cured at different time-points after treatment and none of them relapsed during the follow-up period. In 16 out of 17 patients (94.1%) the PCR turned negative (Fig. 1) during the first follow-up evaluation
281
Fig. 1. PCR amplified products from the conserved region of Leishmania minicircle kDNA by polymerase chain reaction. NC: negative control (no DNA); lanes 1–3: blood samples from patrients with diagnosis of VL before treatment; lanes 4–10: blood samples from patients during the follow-up controls; lane 11: negative control (healthy individual); lane 12: positive PCR control (Leishmania DNA).
that comprised the period of 30 days treatment until 7 days after treatment. Clearance of kDNA occurred before or concomitant to clinical definition of cure, based on absence of fever, normal physical examination and normal blood cells count (bold figures in Table 1). Three patients (ESM-number 5; RAC-number 13 and TMC-number 17) out of eight from whom samples were obtained during the first 15 days of treatment were still positive PCR at 12, 14 and 14 days of treatment, respectively, but kDNA was undetectable in blood samples taken at days 36, 33 and 29 after
Table 1 Leishmania kDNA detected by PCR in peripheral blood seventeen patients with visceral leishmaniasis during and after specific chemotherapy
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Patients
Sex
Age
During and until 7 days after treatment
50–90 days after treatment
>90 days after treatment
ACCS AAOD DO ESVS ESM EFAM GFG JAP JNSR MLK PCS RMM RAC SGS SAD TCAF TMC
F F M M M F F M M F F M M F M F F
27 36 41 22 22 14 21 43 42 45 14 08 34 01 15 06 04
N (26) P (30) N (14) P (23) N (37) N (30) P (12) N (36) N (33) N (7) N (33) N (30) N (37) N (7) N (35) N (28) N (29) N (9) N (30) P (14) N (33) N (11) N (25) N (35), N (33) P (14) N (29)
N (54) P (82) N (56) N (85) N (69) ND N (62) N (68) N (67) N (62) N (92) N (61) N (63) N (65) N (50) N (61) N (83)
N (162) P (112) P (145) N (226) N (142) N (219) ND ND N (153) N (166) ND ND N (183) N (97) N (183) N (109) N (106) ND N (114)
*Day 0 refers to the first day of treatment, P = positive PCR, N = negative PCR, ND = not done. Figures in parenthesis correspond to precise day of blood collection and figures in bold correspond to the day on which cure was clinically defined.
282
J. Disch et al. / Acta Tropica 92 (2004) 279–283
treatment evaluation. Fluctuation of circulating DNA was observed in one patient (DO-number 3) that presented negative PCR at the 14th day of treatment, became positive again at day 23 but turned negative at day 37 and remained so up to 142 days of follow-up. PCR became negative in five out of eight patients from whom samples were available during the first 15 days of treatment. Further follow-up of these patients demonstrated that negative results were sustained. Only one patient (AAOD-number 2) scored positive by PCR during the first period of follow-up (30 days). This patient, a 37-year-old woman had a severe VL with hemoglobin of 3.0 g/dL, 1200 leucocytes and 76,000 platelets/mm3 . She had refused blood transfusion due to religion constraints. At the end of the treatment with meglumine antimoniate, a significant clinical improvement was observed and the absence of symptoms and signs was determined at 82 days after treatment. However, Leishmania kDNA could still be amplified from the patient’s blood up to 145 days after treatment initiation. Only 8 months after treatment the PCR turned negative. At the same time, skin test became positive (reaction of 2.2 cm of diameter). In this group of patients, anti-L. chagasi IgG antibody detected by the immunofluorescent assay, repeated at the same follow-up occasions presented a tendency for decreasing titers over the period but remained positive.
4. Discussion In this study, we observed a rapid clearance of Leishmania kDNA in peripheral blood samples during or soon after treatment of VL. A specific test that accurately assures cure and/or predicts recurrence of VL is not yet available and fiercely sought-after. The efficiency of Leishmania DNA detection in peripheral blood of patients with VL may offer a non-invasive highly sensitive tool to help the assessment of cure, although a number of uncertainties are still to be addressed. For instance, is the early presence of DNA a direct evidence of viable pathogens or could this evidence be puzzled by the amplification of intact free nucleic acids? Two previous reports evaluated the presence of Leishmania DNA as a cure assessment tool in immunocompetent patients. Nuzum et al. (1995) evaluated 13
Indian patients in whom drug treatment had apparently been successful by clinical criteria and observed 14 and 0% of blood PCR positive patients at 1 and 6 months after treatment, respectively. Cascio et al. (2002) observed that Leishmania DNA was cleared from the peripheral blood within a median of 6 days after the start of lipossomal anfotericin and 14 days after the start of meglumine antimoniate in ten Italian children with VL. In this study, that included repeated follow-up of treated patients, 94% (16/17) of the patients with positive PCR at pre-treatment evaluation became negative during the 30-day treatment period or within the first week after treatment (37 days). Further followup of these patients showed that negative results were sustained. It is not clear to us why one patient had detectable Leishmania DNA until a long time after clinical cure was attained. This case differs from the observed by Cascio et al. (2002), that followed a patient that persisted with positive blood PCR after 3 months after treatment but relapsed 15 days latter. These observations suggest that the persistence of parasite DNA in blood (as well the persistence of parasites in bone marrow) seem not to be an unequivocal indicator of further relapse that is multi-factorial determined. As all of these patients were successfully treated and none of them relapsed, a broad and definitive conclusion about the utility of the blood PCR on the prediction of cure and relapse was not possible. Only the accumulation of experience with this tool will provide this information. This low frequency of relapses or treatment failure and the rapid clearance of parasite DNA in immunocompetents diverge from the evolution of patients co-infected with HIV, which present lower rates of cure, higher frequency of relapses and frequent persistence of circulating DNA. For instance, Fisa et al. (2002) using a nested PCR detected Leishmania infantum genomic DNA, 1 month after treatment in 7 of 10 patients, eight of them infected with HIV. All five that relapsed belonged to the HIV-infected group. Through this study-protocol, it was possible to conclude that the clearance of Leishmania kDNA from peripheral blood of patients with visceral leishmaniasis hints to occur during or shortly after treatment, concurring or preceding clinical recovery. Our data reinforce that blood PCR constitutes a useful non-invasive method for monitoring the cure of visceral leishmaniasis.
J. Disch et al. / Acta Tropica 92 (2004) 279–283
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