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Serological signatures of clinical cure following successful treatment with sodium stibogluconate in Ethiopian visceral leishmaniasis
Cytokine 91 (2017) 6–9
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Serological signatures of clinical cure following successful treatment with sodium stibogluconate in Ethiopian visceral leishmaniasis Endalamaw Gadisa a,⇑, Geremew Tasew a,b, Adugna Abera a, Woyneshet Gelaye c, Menberework Chanyalew a, Markos Abebe a, Tamás Laskay d, Abraham Aseffa a a
Armauer Hansen Research Institute, Addis Ababa, Ethiopia Ethiopian Public Health Institute, Addis Ababa, Ethiopia Amhara Regional State Referral and Research Laboratory, Bahir Dar, Ethiopia d University of Lübeck, Lübeck, Germany b c
a r t i c l e
i n f o
Article history: Received 4 July 2016 Received in revised form 15 November 2016 Accepted 29 November 2016
Keywords: Signature biomarker Visceral leishmaniasis treatment Soluble CD40 ligand Matrix metalloproteinase nine Interleukin ten
a b s t r a c t Background: In Ethiopia, visceral leishmaniasis (VL) is a growing public health threat. Among the key challenges in VL control in Ethiopia is lack of an effective test of cure. The recommended test of cure is parasite detection. As sterile cure is not expected with the current widely used drugs, the value of parasite detection as test of cure is questionable. Moreover, the sampling is invasive, requires a well-equipped facility and highly skilled personnel, which are all hardly found in endemic set-ups. Objective: Our aim was to assess the value of sCD40L, MMP9 and IL-10 serum levels as signature biomarkers of clinical cure in VL cases from Ethiopia. Methods: A total of 45 VL cases before and after treatment and 30 endemic healthy controls were included in the study. Sandwich ELISA was used to measure serum levels of sCD40L, MMP9 and IL-10. Result: The mean sCD40L, MMP9 and IL-10 serum levels changed significantly at clinical cure. At individual case level sCD40L and MMP9 showed an increasing trend. Yet, the degree of increase in serum level of MMP9 seems to be affected by nutritional status of the individual VL case. The mean IL-10 serum level was significantly reduced at clinical cure. As seen on case by case basis, all demonstrated a declining trend except that two VL cases had a high IL10 level at clinical cure. Conclusion: Our result is suggestive of the possibility of developing a signature biomarker to monitor VL treatment in Ethiopia using one or a combination of parameters. Ó 2016 Published by Elsevier Ltd.
1. Background Visceral leishmaniasis (VL or Kala-azar) always has a fatal course without early diagnosis and proper treatment; even up to 20% of those on treatment could die [1–4]. East African VL foci contribute for 29,400–56,700 of the estimated 202,200–389,100 global incidences [5]. And VL is the cause of at least 40,000 deaths annually, and a loss of approximately 385,000 disability-adjusted life years [5,6]. Despite the global momentum for control/elimination [7], VL remains a growing public health concern in East Africa. As per the World Health Organization 2015 expert report, the key challenges in VL control in the region are inconsistent performance ⇑ Corresponding author at: Armauer Hansen Research Institute, P.O. Box 1005, Jimma Road, Addis Ababa, Ethiopia. E-mail addresses: [email protected] (E. Gadisa), getas73@yahoo. com (G. Tasew), [email protected] (A. Abera), [email protected] (W. Gelaye), [email protected] (M. Chanyalew), [email protected] (M. Abebe), [email protected] (T. Laskay), [email protected] (A. Aseffa). http://dx.doi.org/10.1016/j.cyto.2016.11.016 1043-4666/Ó 2016 Published by Elsevier Ltd.
of rapid diagnostic tests (RDTs); lack of drug resistance monitoring; insufficient access to treatment and lack of test of cure [7]. Parasite detection is the recommended test of cure in the Ethiopian guideline. This involves an invasive procedure of spleen or bone marrow aspirates. The procedure needs highly skilled personnel with well-equipped facility, which is not found in most of endemic site health care facilities. Thus, availing tools for assessing therapeutic success is a high priority task. Both the innate and adaptive arm of the immune system were shown to play an important role in VL. Neutrophils that express MMP9 and inflammatory monocytes are important in the splenic tissue remodelling that occurs during clinical VL [8]. Moreover, it has been documented that the serum profile of MMP9 is inversely correlated with spleen size and parasite load in VL [9]. In experimental Leishmania infection, a healing phenotype was correlated with CD40L-dependent T-cell response primed by DCs at the site of infection [10]. Serum sCD40L level was reported to negatively correlate with spleen size and parasite load in VL [9]. Moreover,
7
E. Gadisa et al. / Cytokine 91 (2017) 6–9
sCD40L from VL exposed individuals was shown to help control Leishmania infection in macrophages in vitro [11]. Among the cytokines demonstrated to correlate with VL progression and recovery is IL-10. Its profiles were shown to directly correlate with the parasite load [12,13] under various geographic and transmission settings. Thus, our aim was to evaluate the value of MMP9, sCD40L and IL-10 as signature biomarkers of clinical cure in Ethiopian VL patients. 2. Participants and methodology 2.1. Ethical consideration This work was approved by the Armauer Hansen Research Institute and Ethiopian National research ethics review committees, as part of the DFG funded Germen-Africa cooperation project entitled ‘‘Innate immunity in leishmaniasis”. All participants gave written informed consent before enrolment. 2.2. Study design This is a case control study involving 49 consecutive VL cases from Addis Zemen treatment center and 30 endemic health controls from Bura kebele, Libo Kemkem district in Ethiopia. 2.3. Inclusion criteria Confirmed VL cases were included in the study. Diagnosis and clinical cure definition were as per the Ethiopian guideline. Suspected clinical VL cases with fever for more than two weeks, splenomegaly, and/or lymphadenopathy, while living in a known VL endemic area or having travelled to an endemic area were approached. Laboratory confirmation was done with rK39 and DAT. Clinical cure was defined as absence of clinical features of VL after completion of the recommended dose (20 mg/kg/day) and duration of treatment (30 days) with sodium stibogluconate (Generic SSG, Albert David Ltd, Calcutta). Endemic healthy controls (EHC) were same sex and age ±5 years as the index case, had no treatment history for VL, and tested DAT negative. Both study subjects (VL cases) and EHC tested negative for HIV-1/2 serological test performed according to the national test algorithm. 2.4. Exclusion criteria VL cases with HIV co-infection and other known co morbidities. EHC with history of treatment for VL and tested DAT positive. 2.5. Data collection and analysis Sociodemographic, anthropometric and treatment outcome data were documented using a pretested questionnaire. Ten milliliter of venous blood sample was collected before and after treatment from cases. The same volume of blood was collected from the EHC after physical examination, and tested for DAT and HIV1/2. DAT was done as per the protocol of the supplier of the kit, Institute of Tropical medicine (ITMA-DAT/VL, Antwerp, Belgium). Titters 61:400 were considered negative. The serum was stored at 20 °C until use. The level of MMP9, sCD40L and IL-10 was assessed by sandwich ELISA as per the manufacturer’s protocol (DuoSetÒ ELISA development systems, R&D systemsÒ, Minneapolis, USA). Body mass index (BMI, weight (kg)/height (m)2) was used to determine nutritional status. The classification was as per WHO recommendation (http://apps.who.int/bmi). Data analysis was done using GraphPad Prism version 5.00 for Windows (GraphPad
Software, San Diego CA, USA) and STATA version 11 (StataCorp LP, College Station, Texas, USA). Results were considered statistically significant when paired T-test p-value <0.05. 3. Results Forty-nine VL cases, all male and returning from daily labor work in the lowlands of Metema-Humera, the highest burden focus of VL in Ethiopia, were investigated. All participants were negative for HIV. Four cases were found to be malaria co-infected and were thus excluded from the study. Almost all participants (91.2%) were undernourished (Table 1). While 30% (9/30) of the endemic healthy controls had grade 1 thinness; the rest were within the normal range. Serum MMP9, sCD40L and IL-10 levels are altered at clinical cure. The mean serum level of MMP9 at clinical cure (91656.61 pg/ll) showed a significant increase (66412.99 pg/ll, P < 0.0001, t45 = 5.7740) as compared to that before treatment (25243.62 pg/ll). Yet, when cases were clinically cured the mean level of MMP9 was lower by 345,241 pg/ll (P = 0.001, t29 = 9.4048) as compared to the EHCs (Fig. 1A). Moreover, the observed MMP9 serum level seems to be affected by the nutritional status of the cases (Fig. 2). Similarly, the mean serum level of sCD40L at clinical cure was 703.4 pg/ll as opposed to that before treatment, 205.6 pg/ll; a statistically significant increase of 499.95 pg/ll (t45 = 12.67, P < 0.0001). Moreover, the mean serum level of sCD40L was similar to that of endemic healthy controls at clinical cure (P = 0.239) (Fig. 1B). The measured mean serum IL-10 level showed a significant decrease of 409.464 pg/ll (P = 0.001, t45 = 6.8019) by clinical cure (209.6524 pg/ll) as compared to the before treatment (619.1162 pg/ll). And the level was equivalent (P = 0.8064) to the EHCs (Fig. 1C). Considering each individual VL cases and comparing the before and after treatment serum levels; MMP9 (Fig. 3A) and sCD40L (Fig. 3B) showed an increasing trend while IL10 showed a decreasing trend (Fig. 3C) except in two cases where an increased IL-10 was noted. 4. Discussion Visceral leishmaniasis (VL or Kala-azar) in Ethiopia is an increasing public health concern despite the strong government commitment and control effort [14,15]. Regardless of their considerable side effects and/or high cost, drugs remain the mainstay in
Table 1 Clinical and nutritional status of investigated visceral leishmaniasis patients, Addis Zemen Health Center, 2015. Serum level of MMP9, sCD40L and IL-10 before and after treatment of VL cases, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases. Socio demographic data
Number (percentage)
Sex Male
45 (100)
Clinical findings Splenomegaly Hepatosplenomegaly Skin pallor Bleeding tendency Pitting edema
40 (88.9) 5 (11.1) 34 (75.6) 20 (44.4) 14 (31.1)
Nutritional status Grade 3 thinness Grade 2 thinness Grade 1 thinness Normal range
16 (35.6) 13 (28.9) 12 (26.7) 4 (8.9)
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E. Gadisa et al. / Cytokine 91 (2017) 6–9
Fig. 1. The change in mean serum level of MMP 9, sCD40L and IL-10 at clinical cure as compared to the before treatment level and compared to healthy endemic controls, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases, EHC = Endemic healthy controls.
Fig. 2. The change in MMP9 serum level at clinical cure in VL cases in relation to their enrolment serum level and nutritional status, Addis Zemen Health Center, 2015. BT. VL = Before treatment VL cases, and AT.VL = After treatment VL cases; the arrow BT.VL/AT.VL stands for the direction and magnitude of change from the starting level dot BT. VL.
Fig. 3. The trend and direction of change in the serum level of MMP 9, sCD40L and IL-10 considering individual VL case before and after treatment, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases.
VL control. The aim of VL treatment, at least with the current widely used antileishmanial drugs, is to reduce the parasite burden and help the recuperation of the immune response to deal with parasite clearance [16,17]. Thus, the use of parasite detection as signature of treatment efficacy [14] has less relevance. In addition, the sampling is invasive that need well-trained personnel and equipped facilities, which are hardly, found in most Ethiopian
treatment centers. Thus, alternative tests of cure with less invasive sampling that allow analyses of several samples within a short time are needed. Previously reported promising markers that correlated with the amount of circulating Leishmania DNA/parasite burden and showed that these are changed by treatment were assessed in our study [9,13]. Similar to previous observation, serum level of MMP9 increased significantly at clinical cure as
E. Gadisa et al. / Cytokine 91 (2017) 6–9
compared to levels before treatment [9]. Yet, the level after treatment does not reach the level observed in endemic healthy controls, which might be due to the high proportion (90.48%) of undernourishment observed in the VL cases. Though our sample size was not calculated to detect such associations, our observation corroborates reports from animal models which documented that protein energy malnutrition reduced MMP9 production [18]. We observed significantly high sCD40L in sera of patients after treatment as compared to that before treatment. A level comparable to that of the endemic healthy controls was attained at clinical cure. The trend of increase in the serum level of sCD40L with successful treatment was in-line with previous reports [13]. Similar to our finding, a low level of sCD40L was observed in sera from Brazilian VL cases as compared to endemic healthy controls [9]. Moreover, sCD40L from asymptomatically infected individuals was shown to limit the growth of L. infantum [19] in macrophages, further strengthening the evidence of a protective role that sCD40L might have in VL. Thus, our data support previous suggestions that the reduced levels of sCD40L during clinical VL may be a function of parasite-induced perturbation of immune functions. Mixed Th1/ Th2 responses are observed in VL patients in different geographic settings and causative species [20,21]. High level of IL-10 was shown to be a hallmark of clinical VL that declines upon successful treatment [20,21]. Moreover, the level of IL-10 in circulation was shown to correlate with parasite load [12,13]. In agreement with these observations, we saw significant reduction in the serum level of IL-10 at clinical cure when compared to the level observed before treatment. The level of serum IL-10 after treatment was comparable to that of healthy endemic controls. It has also been documented that IL-10 is among those mediators that reduce CD40L expression [22]. Thus, the observed decline of IL-10 and increase of sCD40L with clinical cure might have something to do with this regulatory role of IL-10. The advantage, in addition to their correlation with clinical cure, the usefulness of these cytokines is that they allow for an easier sample collection and analysis at less cost and technical competence. Splenic or bone marrow puncture for test of cure based on parasite detection needs experienced pathologists and laboratory technicians with a well-equipped facility. On the other hand, the sample for using cytokine markers is venous blood followed by an ELISA test that could itself be partially or fully automated expediting both sample collection and analysis with relatively less time, cost and trauma to the patients” As per our data, sCD40L, MMP9 and IL-10 could possibly serve as serological signatures to monitor VL treatment in Ethiopia. However, the effect of protein energy malnutrition on MMP9 and how the markers behave in concomitant infections deserves further evaluation. 5. Conclusion Our data corroborate previous reports by others and indicate the possibility to develop signature prognostic biomarker for VL treatment. We recommend additional validation studies to evaluate how the markers behave in different nutritional status groups and relevant comorbidities prevalent in Ethiopian VL foci. Moreover, rigorous documentation of clinical and biochemical parameters during treatment, including during the 6 months of followup after successful treatment, is highly recommended. This might help in the selection of the best marker and/or combination to monitor treatment as well as optimize required nutritional supplementation.
9
Acknowledgement This work was supported by the German Research Foundation, Germany (DFG, LA 1267-3-1); Innate Immunity in Leishmaniasis Project. We acknowledge the EETB-RTP program (grant number TW009127) of Emory University, United States. References [1] S. Collin, R. Davidson, K. Ritmeijer, K. Keus, Y. Melaku, S. Kipngetich, et al., Conflict and kala-azar: determinants of adverse outcomes of kala-azar among patients in southern Sudan, Clin. Infect. Dis.: Off. Publ. Infect. Dis. Soc. Am. 38 (2004) 612–619. [2] M. Herrero, G. Orfanos, D. Argaw, A. Mulugeta, P. Aparicio, F. Parreno, et al., Natural history of a visceral leishmaniasis outbreak in highland Ethiopia, Am. J. Trop. Med. Hyg. 81 (2009) 373–377. [3] S. Lyons, H. Veeken, J. Long, Visceral leishmaniasis and HIV in Tigray, Ethiopia, Trop. Med. Int. Health: TM & IH 8 (2003) 733–739. [4] T. Haile, S.D. Anderson, Visceral leishmaniasis in northern Ethiopia, East Afr. Med. J. 83 (2006) 389–392. [5] J. Alvar, I.D. Velez, C. Bern, M. Herrero, P. Desjeux, J. Cano, et al., Leishmaniasis worldwide and global estimates of its incidence, PLoS ONE 7 (2012) e35671. [6] R. Reithinger, S. Brooker, J.H. Kolaczinski, Visceral leishmaniasis in eastern Africa – current status, Trans. R. Soc. Trop. Med. Hyg. 101 (2007) 1169–1170. [7] WHO, Visceral Leishmaniasis: Control Strategies and Epidemiological Situation Update in East Africa, Report of a WHO Bi-regional Consultation, Addis Ababa, Ethiopia, 9–11 March 2015, WHO Library Cataloguing-in-Publication Data, World Health Organization, Geneva, 2015. [8] P. Yurdakul, J. Dalton, L. Beattie, N. Brown, S. Erguven, A. Maroof, et al., Compartment-specific remodeling of splenic micro-architecture during experimental visceral leishmaniasis, Am. J. Pathol. 179 (2011) 23–29. [9] F.A. de Oliveira, C. Vanessa Oliveira Silva, N.P. Damascena, R.O. Passos, M.S. Duthie, J.A. Guderian, et al., High levels of soluble CD40 ligand and matrix metalloproteinase-9 in serum are associated with favorable clinical evolution in human visceral leishmaniasis, BMC Infect. Dis. 13 (2013) 331. [10] M.A. Marovich, M.A. McDowell, E.K. Thomas, T.B. Nutman, IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/ CD40 ligand-dependent process, J. Immunol. 164 (2000) 5858–5865. [11] F.A. Oliveira, A.S. Barreto, L.G. Bomfim, T.R. Leite, P.L. Dos Santos, R.P. Almeida, et al., Soluble CD40 ligand in sera of subjects exposed to leishmania infantum infection reduces the parasite load in macrophages, PLoS ONE 10 (2015) e0141265. [12] S. Verma, R. Kumar, G.K. Katara, L.C. Singh, N.S. Negi, V. Ramesh, et al., Quantification of parasite load in clinical samples of leishmaniasis patients: IL10 level correlates with parasite load in visceral leishmaniasis, PLoS ONE 5 (2010) e10107. [13] M.S. Duthie, J. Guderian, A. Vallur, A. Bhatia, P. Lima dos Santos, E. Vieira de Melo, et al., Alteration of the serum biomarker profiles of visceral leishmaniasis during treatment, Eur. J. Clin. Microbiol. Infect. Dis.: Off. Publ. Eur. Soc. Clin. Microbiol. 33 (2014) 639–649. [14] FMoH, in: N. Unit (Ed.), Guideline for Diagnosis, Treatment and Prevention of Leishmaniasis in Ethiopia, second ed., Ethiopian Federal Ministry of Health, Addis Ababa; 2013. [15] E. Gadisa, T. Tsegaw, A. Abera, D.E. Elnaiem, M. den Boer, A. Aseffa, et al., Ecoepidemiology of visceral leishmaniasis in Ethiopia, Paras. Vect. 8 (2015) 381. [16] J.Q. Reimao, J.C. Oliveira, C.T. Trinconi, P.C. Cotrim, A.C. Coelho, S.R. Uliana, Generation of luciferase-expressing Leishmania infantum chagasi and assessment of miltefosine efficacy in infected hamsters through bioimaging, PLoS Negl. Trop. Dis. 9 (2015) e0003556. [17] M. Sudarshan, J.L. Weirather, M.E. Wilson, S. Sundar, Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis, J. Antimicrob. Chemother. 66 (2011) 1751–1755. [18] L.D. Kozaci, M. Balkaya, C. Unsal, H. Unsal, F. Kargin, I.C. Chikanza, The expression of matrix metalloproteinases 8 and 9 by neutrophils of Wistar albino rats with severe qualitative and quantitative protein malnutrition, Int. J. Vitam. Nutr. Res. 75 (2005) 257–265. [19] F.A. de Oliveira, A.S. Barreto, L.G. Bomfim, T.R. Leite, P.L. Dos Santos, R.P. de Almeida, et al., Soluble CD40 ligand in sera of subjects exposed to Leishmania infantum infection reduces the parasite load in macrophages, PLoS ONE 10 (2015) e0141265. [20] A. Caldas, C. Favali, D. Aquino, V. Vinhas, J. van Weyenbergh, C. Brodskyn, et al., Balance of IL-10 and interferon-gamma plasma levels in human visceral leishmaniasis: implications in the pathogenesis, BMC Infect. Dis. 5 (2005) 113. [21] A. Khoshdel, A. Alborzi, M. Rosouli, E. Taheri, S. Kiany, M.H. Javadian, Increased levels of IL-10, IL-12, and IFN- in patients with visceral leishmaniasis, Braz. J. Infect. Dis. 13 (2009) 44–46. [22] D. Daoussis, A.P. Andonopoulos, S.N. Liossis, Targeting CD40L: a promising therapeutic approach, Clin. Diagn. Lab. Immunol. 11 (2004) 635–641.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.journals.elsevier.com/cytokine
Serological signatures of clinical cure following successful treatment with sodium stibogluconate in Ethiopian visceral leishmaniasis Endalamaw Gadisa a,⇑, Geremew Tasew a,b, Adugna Abera a, Woyneshet Gelaye c, Menberework Chanyalew a, Markos Abebe a, Tamás Laskay d, Abraham Aseffa a a
Armauer Hansen Research Institute, Addis Ababa, Ethiopia Ethiopian Public Health Institute, Addis Ababa, Ethiopia Amhara Regional State Referral and Research Laboratory, Bahir Dar, Ethiopia d University of Lübeck, Lübeck, Germany b c
a r t i c l e
i n f o
Article history: Received 4 July 2016 Received in revised form 15 November 2016 Accepted 29 November 2016
Keywords: Signature biomarker Visceral leishmaniasis treatment Soluble CD40 ligand Matrix metalloproteinase nine Interleukin ten
a b s t r a c t Background: In Ethiopia, visceral leishmaniasis (VL) is a growing public health threat. Among the key challenges in VL control in Ethiopia is lack of an effective test of cure. The recommended test of cure is parasite detection. As sterile cure is not expected with the current widely used drugs, the value of parasite detection as test of cure is questionable. Moreover, the sampling is invasive, requires a well-equipped facility and highly skilled personnel, which are all hardly found in endemic set-ups. Objective: Our aim was to assess the value of sCD40L, MMP9 and IL-10 serum levels as signature biomarkers of clinical cure in VL cases from Ethiopia. Methods: A total of 45 VL cases before and after treatment and 30 endemic healthy controls were included in the study. Sandwich ELISA was used to measure serum levels of sCD40L, MMP9 and IL-10. Result: The mean sCD40L, MMP9 and IL-10 serum levels changed significantly at clinical cure. At individual case level sCD40L and MMP9 showed an increasing trend. Yet, the degree of increase in serum level of MMP9 seems to be affected by nutritional status of the individual VL case. The mean IL-10 serum level was significantly reduced at clinical cure. As seen on case by case basis, all demonstrated a declining trend except that two VL cases had a high IL10 level at clinical cure. Conclusion: Our result is suggestive of the possibility of developing a signature biomarker to monitor VL treatment in Ethiopia using one or a combination of parameters. Ó 2016 Published by Elsevier Ltd.
1. Background Visceral leishmaniasis (VL or Kala-azar) always has a fatal course without early diagnosis and proper treatment; even up to 20% of those on treatment could die [1–4]. East African VL foci contribute for 29,400–56,700 of the estimated 202,200–389,100 global incidences [5]. And VL is the cause of at least 40,000 deaths annually, and a loss of approximately 385,000 disability-adjusted life years [5,6]. Despite the global momentum for control/elimination [7], VL remains a growing public health concern in East Africa. As per the World Health Organization 2015 expert report, the key challenges in VL control in the region are inconsistent performance ⇑ Corresponding author at: Armauer Hansen Research Institute, P.O. Box 1005, Jimma Road, Addis Ababa, Ethiopia. E-mail addresses: [email protected] (E. Gadisa), getas73@yahoo. com (G. Tasew), [email protected] (A. Abera), [email protected] (W. Gelaye), [email protected] (M. Chanyalew), [email protected] (M. Abebe), [email protected] (T. Laskay), [email protected] (A. Aseffa). http://dx.doi.org/10.1016/j.cyto.2016.11.016 1043-4666/Ó 2016 Published by Elsevier Ltd.
of rapid diagnostic tests (RDTs); lack of drug resistance monitoring; insufficient access to treatment and lack of test of cure [7]. Parasite detection is the recommended test of cure in the Ethiopian guideline. This involves an invasive procedure of spleen or bone marrow aspirates. The procedure needs highly skilled personnel with well-equipped facility, which is not found in most of endemic site health care facilities. Thus, availing tools for assessing therapeutic success is a high priority task. Both the innate and adaptive arm of the immune system were shown to play an important role in VL. Neutrophils that express MMP9 and inflammatory monocytes are important in the splenic tissue remodelling that occurs during clinical VL [8]. Moreover, it has been documented that the serum profile of MMP9 is inversely correlated with spleen size and parasite load in VL [9]. In experimental Leishmania infection, a healing phenotype was correlated with CD40L-dependent T-cell response primed by DCs at the site of infection [10]. Serum sCD40L level was reported to negatively correlate with spleen size and parasite load in VL [9]. Moreover,
7
E. Gadisa et al. / Cytokine 91 (2017) 6–9
sCD40L from VL exposed individuals was shown to help control Leishmania infection in macrophages in vitro [11]. Among the cytokines demonstrated to correlate with VL progression and recovery is IL-10. Its profiles were shown to directly correlate with the parasite load [12,13] under various geographic and transmission settings. Thus, our aim was to evaluate the value of MMP9, sCD40L and IL-10 as signature biomarkers of clinical cure in Ethiopian VL patients. 2. Participants and methodology 2.1. Ethical consideration This work was approved by the Armauer Hansen Research Institute and Ethiopian National research ethics review committees, as part of the DFG funded Germen-Africa cooperation project entitled ‘‘Innate immunity in leishmaniasis”. All participants gave written informed consent before enrolment. 2.2. Study design This is a case control study involving 49 consecutive VL cases from Addis Zemen treatment center and 30 endemic health controls from Bura kebele, Libo Kemkem district in Ethiopia. 2.3. Inclusion criteria Confirmed VL cases were included in the study. Diagnosis and clinical cure definition were as per the Ethiopian guideline. Suspected clinical VL cases with fever for more than two weeks, splenomegaly, and/or lymphadenopathy, while living in a known VL endemic area or having travelled to an endemic area were approached. Laboratory confirmation was done with rK39 and DAT. Clinical cure was defined as absence of clinical features of VL after completion of the recommended dose (20 mg/kg/day) and duration of treatment (30 days) with sodium stibogluconate (Generic SSG, Albert David Ltd, Calcutta). Endemic healthy controls (EHC) were same sex and age ±5 years as the index case, had no treatment history for VL, and tested DAT negative. Both study subjects (VL cases) and EHC tested negative for HIV-1/2 serological test performed according to the national test algorithm. 2.4. Exclusion criteria VL cases with HIV co-infection and other known co morbidities. EHC with history of treatment for VL and tested DAT positive. 2.5. Data collection and analysis Sociodemographic, anthropometric and treatment outcome data were documented using a pretested questionnaire. Ten milliliter of venous blood sample was collected before and after treatment from cases. The same volume of blood was collected from the EHC after physical examination, and tested for DAT and HIV1/2. DAT was done as per the protocol of the supplier of the kit, Institute of Tropical medicine (ITMA-DAT/VL, Antwerp, Belgium). Titters 61:400 were considered negative. The serum was stored at 20 °C until use. The level of MMP9, sCD40L and IL-10 was assessed by sandwich ELISA as per the manufacturer’s protocol (DuoSetÒ ELISA development systems, R&D systemsÒ, Minneapolis, USA). Body mass index (BMI, weight (kg)/height (m)2) was used to determine nutritional status. The classification was as per WHO recommendation (http://apps.who.int/bmi). Data analysis was done using GraphPad Prism version 5.00 for Windows (GraphPad
Software, San Diego CA, USA) and STATA version 11 (StataCorp LP, College Station, Texas, USA). Results were considered statistically significant when paired T-test p-value <0.05. 3. Results Forty-nine VL cases, all male and returning from daily labor work in the lowlands of Metema-Humera, the highest burden focus of VL in Ethiopia, were investigated. All participants were negative for HIV. Four cases were found to be malaria co-infected and were thus excluded from the study. Almost all participants (91.2%) were undernourished (Table 1). While 30% (9/30) of the endemic healthy controls had grade 1 thinness; the rest were within the normal range. Serum MMP9, sCD40L and IL-10 levels are altered at clinical cure. The mean serum level of MMP9 at clinical cure (91656.61 pg/ll) showed a significant increase (66412.99 pg/ll, P < 0.0001, t45 = 5.7740) as compared to that before treatment (25243.62 pg/ll). Yet, when cases were clinically cured the mean level of MMP9 was lower by 345,241 pg/ll (P = 0.001, t29 = 9.4048) as compared to the EHCs (Fig. 1A). Moreover, the observed MMP9 serum level seems to be affected by the nutritional status of the cases (Fig. 2). Similarly, the mean serum level of sCD40L at clinical cure was 703.4 pg/ll as opposed to that before treatment, 205.6 pg/ll; a statistically significant increase of 499.95 pg/ll (t45 = 12.67, P < 0.0001). Moreover, the mean serum level of sCD40L was similar to that of endemic healthy controls at clinical cure (P = 0.239) (Fig. 1B). The measured mean serum IL-10 level showed a significant decrease of 409.464 pg/ll (P = 0.001, t45 = 6.8019) by clinical cure (209.6524 pg/ll) as compared to the before treatment (619.1162 pg/ll). And the level was equivalent (P = 0.8064) to the EHCs (Fig. 1C). Considering each individual VL cases and comparing the before and after treatment serum levels; MMP9 (Fig. 3A) and sCD40L (Fig. 3B) showed an increasing trend while IL10 showed a decreasing trend (Fig. 3C) except in two cases where an increased IL-10 was noted. 4. Discussion Visceral leishmaniasis (VL or Kala-azar) in Ethiopia is an increasing public health concern despite the strong government commitment and control effort [14,15]. Regardless of their considerable side effects and/or high cost, drugs remain the mainstay in
Table 1 Clinical and nutritional status of investigated visceral leishmaniasis patients, Addis Zemen Health Center, 2015. Serum level of MMP9, sCD40L and IL-10 before and after treatment of VL cases, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases. Socio demographic data
Number (percentage)
Sex Male
45 (100)
Clinical findings Splenomegaly Hepatosplenomegaly Skin pallor Bleeding tendency Pitting edema
40 (88.9) 5 (11.1) 34 (75.6) 20 (44.4) 14 (31.1)
Nutritional status Grade 3 thinness Grade 2 thinness Grade 1 thinness Normal range
16 (35.6) 13 (28.9) 12 (26.7) 4 (8.9)
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Fig. 1. The change in mean serum level of MMP 9, sCD40L and IL-10 at clinical cure as compared to the before treatment level and compared to healthy endemic controls, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases, EHC = Endemic healthy controls.
Fig. 2. The change in MMP9 serum level at clinical cure in VL cases in relation to their enrolment serum level and nutritional status, Addis Zemen Health Center, 2015. BT. VL = Before treatment VL cases, and AT.VL = After treatment VL cases; the arrow BT.VL/AT.VL stands for the direction and magnitude of change from the starting level dot BT. VL.
Fig. 3. The trend and direction of change in the serum level of MMP 9, sCD40L and IL-10 considering individual VL case before and after treatment, Addis Zemen Health Center, 2015. BT.VL = Before treatment VL cases, and AT.VL = After treatment VL cases.
VL control. The aim of VL treatment, at least with the current widely used antileishmanial drugs, is to reduce the parasite burden and help the recuperation of the immune response to deal with parasite clearance [16,17]. Thus, the use of parasite detection as signature of treatment efficacy [14] has less relevance. In addition, the sampling is invasive that need well-trained personnel and equipped facilities, which are hardly, found in most Ethiopian
treatment centers. Thus, alternative tests of cure with less invasive sampling that allow analyses of several samples within a short time are needed. Previously reported promising markers that correlated with the amount of circulating Leishmania DNA/parasite burden and showed that these are changed by treatment were assessed in our study [9,13]. Similar to previous observation, serum level of MMP9 increased significantly at clinical cure as
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compared to levels before treatment [9]. Yet, the level after treatment does not reach the level observed in endemic healthy controls, which might be due to the high proportion (90.48%) of undernourishment observed in the VL cases. Though our sample size was not calculated to detect such associations, our observation corroborates reports from animal models which documented that protein energy malnutrition reduced MMP9 production [18]. We observed significantly high sCD40L in sera of patients after treatment as compared to that before treatment. A level comparable to that of the endemic healthy controls was attained at clinical cure. The trend of increase in the serum level of sCD40L with successful treatment was in-line with previous reports [13]. Similar to our finding, a low level of sCD40L was observed in sera from Brazilian VL cases as compared to endemic healthy controls [9]. Moreover, sCD40L from asymptomatically infected individuals was shown to limit the growth of L. infantum [19] in macrophages, further strengthening the evidence of a protective role that sCD40L might have in VL. Thus, our data support previous suggestions that the reduced levels of sCD40L during clinical VL may be a function of parasite-induced perturbation of immune functions. Mixed Th1/ Th2 responses are observed in VL patients in different geographic settings and causative species [20,21]. High level of IL-10 was shown to be a hallmark of clinical VL that declines upon successful treatment [20,21]. Moreover, the level of IL-10 in circulation was shown to correlate with parasite load [12,13]. In agreement with these observations, we saw significant reduction in the serum level of IL-10 at clinical cure when compared to the level observed before treatment. The level of serum IL-10 after treatment was comparable to that of healthy endemic controls. It has also been documented that IL-10 is among those mediators that reduce CD40L expression [22]. Thus, the observed decline of IL-10 and increase of sCD40L with clinical cure might have something to do with this regulatory role of IL-10. The advantage, in addition to their correlation with clinical cure, the usefulness of these cytokines is that they allow for an easier sample collection and analysis at less cost and technical competence. Splenic or bone marrow puncture for test of cure based on parasite detection needs experienced pathologists and laboratory technicians with a well-equipped facility. On the other hand, the sample for using cytokine markers is venous blood followed by an ELISA test that could itself be partially or fully automated expediting both sample collection and analysis with relatively less time, cost and trauma to the patients” As per our data, sCD40L, MMP9 and IL-10 could possibly serve as serological signatures to monitor VL treatment in Ethiopia. However, the effect of protein energy malnutrition on MMP9 and how the markers behave in concomitant infections deserves further evaluation. 5. Conclusion Our data corroborate previous reports by others and indicate the possibility to develop signature prognostic biomarker for VL treatment. We recommend additional validation studies to evaluate how the markers behave in different nutritional status groups and relevant comorbidities prevalent in Ethiopian VL foci. Moreover, rigorous documentation of clinical and biochemical parameters during treatment, including during the 6 months of followup after successful treatment, is highly recommended. This might help in the selection of the best marker and/or combination to monitor treatment as well as optimize required nutritional supplementation.
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Acknowledgement This work was supported by the German Research Foundation, Germany (DFG, LA 1267-3-1); Innate Immunity in Leishmaniasis Project. We acknowledge the EETB-RTP program (grant number TW009127) of Emory University, United States. References [1] S. Collin, R. Davidson, K. Ritmeijer, K. Keus, Y. Melaku, S. Kipngetich, et al., Conflict and kala-azar: determinants of adverse outcomes of kala-azar among patients in southern Sudan, Clin. Infect. Dis.: Off. Publ. Infect. Dis. Soc. Am. 38 (2004) 612–619. [2] M. Herrero, G. Orfanos, D. Argaw, A. Mulugeta, P. Aparicio, F. Parreno, et al., Natural history of a visceral leishmaniasis outbreak in highland Ethiopia, Am. J. Trop. Med. Hyg. 81 (2009) 373–377. [3] S. Lyons, H. Veeken, J. Long, Visceral leishmaniasis and HIV in Tigray, Ethiopia, Trop. Med. Int. Health: TM & IH 8 (2003) 733–739. [4] T. Haile, S.D. Anderson, Visceral leishmaniasis in northern Ethiopia, East Afr. Med. J. 83 (2006) 389–392. [5] J. Alvar, I.D. Velez, C. Bern, M. 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