Elevated Serum ADA Activity as a Marker for Diagnosis and Prognosis of Visceral Leishmaniasis and Post Kala-Azar Dermal Leishmaniasis in Indian Patients

Serum adenosine deaminase (ADA) activity increases in diseases where cellular immunity is involved. Since cell-mediated immune responses play a paramount role in the pathogenesis and healing of the visceral leishmaniasis, therefore, the present study was undertaken to evaluate the serum ADA activity in different pathological conditions. Adenosine deaminase was determined in sera of active visceral leishmaniasis (VL) patients (n = 39), active postkala-azar dermal leishmaniasis (PKDL) cases (n = 34) at the point of diagnosis and after treatment stages along with healthy controls (n = 30), endemic healthy subjects (n = 34) and endemic asymptomatic subjects (n = 34).Our in-vitro result revealed that monocytes secrete significant ADA level in response to Leishmania donovani (L.donovani) stimulation. The serum ADA activity in active VL and PKDL subjects were found to be significantly higher than that of respective treated cases and healthy controls. We also observed a marginal number (17.6%) of endemic asymptomatic subjects showed elevated serum ADA activity. Further, the ADA activity in PKDL was found to be decreased gradually during the different phases of treatment. Interestingly, 2 out of 32 treated VL cases found to have high serum ADA activity during follow up period were relapsed within few days. These results suggest the possibility of ADA as a marker of clinical pathogenesis and can be used as a surrogate marker in the diagnosis and prognosis of VL and PKDL.


Introduction
The leishmaniases are protozoan parasite disease caused by more than 20 Leishmania species that are transmitted to humans by the bites of infected sandflies. Among these, visceral leishmaniasis (VL) caused by Leishmania donovani (L.donovani), is the most severe in the Indian subcontinent and is fatal if untreated. Post kala-azar dermal leishmaniasis (PKDL) is a dermal complication, caused as a sequel to VL, it is characterised by a macular, maculopapular and nodular rash in a patient who has recovered from VL and who is otherwise well [1]. In India, it manifests in 5-15 per cent of VL cases after months or several years of remission from infection, while in Sudan, it develops within weeks or months in 50-60 per cent of cured VL cases [2,3]. Thus, it is prevalent in the areas where Leishmania donovani is the causative organism [1,4].
Early and accurate diagnosis and treatment remain key factors to curb the VL. In addition, a proper prognostic marker fends off treatment failures. Microscopy remains the gold standard for the diagnosis of VL. Apart from this, rK39 based immuno-chromatographic test (ICT), direct agglutination test (DAT) and PCR have shown high diagnostic accuracy [5][6][7], but these have some inherent limitations. Although the specificity is high, the sensitivity of microscopy varies from organ to organ, spleen has highest sensitivity (93-99%) followed by bone marrow (53-86%) or lymph node (53-65%) aspirates [8,9]. Apart from this, invasive method of sample collection needs the expertise and in some cases spleenic aspiration is fatal [10]. Antibody based immunostrip (rK39) are currently the best available diagnostic tool for VL for use in remote areas. rK39 based test showed sensitivity and specificity estimates of 93.9% and 95.3% respectively [5,6]. However, this test has been shown to be less accurate in East Africa [11]. Moreover, a significant proportion of asymptomatic individuals test positive for the rK39 and the seroprevalence in healthy populations varies from <10% to up to >30% [12,13], therefore, relapse can't be diagnosed by this strip [14][15][16]. DAT has been extensively validated in most endemic areas, with sensitivity and specificity estimates of 94.8 and 97.1 respectively [12]. DAT may yield positive results for a long time after complete cure and thus has not proved to be of much prognostic value [17] and also it requires equipment such as microtitreplate and micropipettes. Although PCR based method gives higher sensitivity (93.80) [7], however, its sensitivity for the detection of Leishmania DNA in the blood of parasitologically proven VL cases was only 70% [18] and is poorly adapted to field settings [19]. In the absence of animal reservoir PKDL patients are thought to be the reservoirs for visceral leishmaniasis in Indian subcontinent and this necessitates an accurate diagnosis of PKDL [20]. Methods for the diagnosing PKDL mainly consists of clinical signs and symptoms and pre-history of VL, parasite demonstration on the skin, leishmanin skin test and immuno-chromatographic test [16]. The clinical signs may often confuse with other skin disorder [21]. Other methods often lack sensitivity or specificity as (i) only 20-40% positive parasite demonstration on the smear because of low parasite load [1] (ii) serological tests may be positive due to the past occurrence of VL (iii) the leishmanin skin test (LST) may or may not be positive. In addition, around 10% of cases have no history of VL, and 10% of cases have no positive serological test making a strict clinical definition important [21].
The assay of ADA activity in the serum and other biologic fluids is very useful for an accurate diagnosis of many pathological conditions [22,23]. Although VL and PKDL are caused by the same parasite but these two diseases differ in many aspect of pathogenesis and disease progression due to their localization in the body [24]. Erel et al. found an increased lymphocytic specific ADA activity in patients with cutaneous leishmaniasis [25]. But reports on sera ADA levels in Indian VL are scarce, one study by Tripati et al showed increased ADA level in Indian VL patients during active VL and subsequent decreased ADA after treatment and another study by A.K.Rai et al was inconclusive as they didn't not measure the serum ADA activity after treatment [26,27]. Most importantly, since symptoms and pathophysiology of VL and PKDL differ considerably [28,29], a study on serum ADA level between VL and PKDL will shed a new light on the diagnosis and better understanding of the pathophysiology of these diseases. Interestingly, a study from Nepal shows that the risk of seroconversion in asymptomatic was higher in Indian than in Nepalese [30]. Therefore, on this context, it is also important to access the serum ADA of asymptomatic subjects because diagnosis of asymptomatic leishmanial infection is hampered by the lack of validated definitions and appropriate biological markers. Here, our study is an attempt to investigate the serum ADA activities of patients with visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and endemic asymptomatic subjects in comparison with healthy control.

Clinical samples and diagnosis
Study groups of persons examined. VL, PKDL, asymptomatic and endemic and nonendemic healthy subjects of both sexes aged from 13 to 65 years were sampled from RMRI OPD, endemic area (District Saran, Bihar, India) and non endemic area (District Patna, Bihar, India). Fresh VL cases were treated with 10 mg/kg body weight single dose Ambisomeandfollowed up for three to six months after treatment. Confirmed PKDL patients were treated by infusion with amphotericin B deoxycholate (0.5 mg/kg body weight/day on days 1-20, 50-70 and 100-120) and followed up for three to six months after third course. The study was started after obtaining their informed and written consent. In case of minor, written consent was obtained from his/her guardian on his/her behalf. Approaching subjects unwilling to give their informed consent or were positive for tuberculosis, malaria, kidney, heart and liver diseases, HIV or with symptoms of asthma or rheumatic arthritis were excluded from this study. Diagnosis was confirmed by demonstration of amastigotes in spleenic/bone marrow aspirate or microscopically. Asymptomatic cases where defined as endemic area individuals with rK39 positive without signs and symptoms.
Group 1. Thirty nine (39) newly diagnosed patients with visceral leishmaniasis. All the active VL patients presented characteristic signs and symptoms of the disease and diagnosis was confirmed by the presence of L.donovani either in Giemsa stained bone marrow aspirate or spleenic aspirate.
Group 3. Thirty four (34) clinically diagnosed PKDL patients of different group (macular/ popular lesions) were included in this study.
Group 4. Twenty nine (29) treated PKDL patients (three courses, every course consists of Amphotericin B deoxycholate 0.5 mg/kg body weight/day for twenty days) with decreased or no macular or popular lesions were considered.
Group 5. Thirty four (34) endemic asymptomatic persons, with positive rK39, apparently without signs and symptoms of the diseases were included. Group 6. Thirty (30) non-endemic healthy subjects, with rK39 negative and without sign and symptoms of the disease and no case history of leishmaniasis.
Group 7. Thirty four (34) endemic healthy subjects with rK39 negative and without any signs and symptoms of the diseases were considered for further study.

Serum collection
Blood samples were collected in plane blood collection vials (Becton Dickinson, San Diego, USA) and let it for room temperature for 30 min and upper serum is transferred to sterile eppendorf tube and centrifuged at 3000 rpm for 10 min at 4°C. 20 μl serum supernatant was used for measurement of ADA activity and rest is stored at -20°Cfor future usage.Blood collected in thefield were transported in ice packs and serum was separated and ADA assay was performed immediately or stored at -20°C and used for ADA assay within a week. For PKDL, blood sample was collected at the start of the treatment and ADA assay was carried out on the same day and rest is stored at -20°C for future usage.

Preparation of cell lysate
Mononuclearcytes and granulocytes were separated by buffy coat method [31]. Cells were resuspended in sterile distilled water and lysed by sonication. Lysate was centrifuged at 8000 rpm for 10 min at 4°C and 50 μg of supernatant protein was used for measurement of ADA activity and rest is stored at -20°Cfor further usage.

ADA activity in monocytes culture supernatant
PBMCs were isolated from five healthy donor and 2 x 10 6 cells per well seeded on six well plate and incubated overnight at 37 0 C CO 2 incubator. Non-adherent cells were washed out and monocytes were pulsed with 1:10 monocytes to metacyclic Leishmania donovani promastigotes for 12-16 hrs. Culture supernatant was collected after 48 hours and used for measurement of ADA activity.

Measurement of ADA activity
ADA activity was measured by ADA-MTB kit (Tulip Diagnostic, India) according to the manufactured protocol.

Statistical analysis
Student's two-tailed paired t test or unpaired t test was used to determine the difference between the groups studied. Statistical analysis was performed using GraphPad Prism 5, USA software and all the data are expressed as Mean ± SEM (standard error of the mean). P<0.05 was considered statistically significant for all the analysis.

Mononuclear cells are the source of serum ADA level during Leishmania donovani infection
In vitro stimulation of monocytes with L.donovani revealed a significant increased ADA activity in culture supernatant as compared to unstimulated/control monocytes (Fig 1, p<0.001). Further, we isolated the mononucleocytes and granulocytes from whole bloods of VL and PKDL in pre and post treatment stage and evaluated the intracellular ADA levels from mononuclear cells and granulocytes from all the subjects. We found significantly higher ADA level in mononuclear cell lysate in VL pre-treatment stages compared to VL post treatment stages and healthy control (Fig 2A, p<0.05), whereas in PKDL cases although active PKDL cases shows high ADA activity compared to treated PKDL cases but difference was not statistically significant (Fig 2A, p>0.05). Further, active VL cases showed significantly higher ADA activity compared to active PKDL, treated PKDL and healthy control (Fig 2A, p<0.05).We could not find any significant difference in ADA activity of granulocytes lysate between these groups ( Fig  2B, p>0.05).

Serum ADA activity is elevated during active VL and PKDL disease
The experiment was designed to evaluate the level of serum ADA activity in active VL and PKDL patients in their pre and post treatment. A significantly increased serum ADA activity was found in active VL and PKDL pre-treatment stages compared to their respective post treatment stages and healthy subjects (Fig 3A, p<0.05). Such higher ADA activity in serum was, however, reduced 3.08 and 2.30 fold following successful treatment of VL and PKDL subject respectively (Fig 3A, p <0.05). However, we observed a significantly higher serum activity in active VL cases compared to active PKDL cases (Fig 3A, p<0.05). Further, the serum ADA activity in PKDL was found to be decreased gradually as the treatment progresses (Fig 3B,  p<0.05).

Asymptomatic subjects shows uneven ADA activity
Since, a marginal number of asymptomatic individuals will convert into VL clinical manifestations [32]. To known the status of serum ADA activity of asymptomatic individuals, we measured the serum ADA activity of endemic asymptomatic subjects and endemic healthy subjects from endemic area. The serum ADA level of endemic healthy subjects found to be in normal range, but the asymptomatic subjects showed a variation (Fig 4), 17.6% (six out of thirty four) asymptomatic subjects found to possess elevated serum ADA level.

Discussion
The ADA activity originates from the action of two principal isoenzymes, ADA-1 and ADA-2, which have different pH, Michaelis constant (Km) and relative substrate specificity patterns   [33]. ADA-1 is present in all tissues and is essential for an efficient immune response whereas ADA-2 is exclusively present in monocytes and macrophages and is secreted in response to pathogen [34,35]. It has been suggested that elevated serum ADA activity reflects the proliferation of T lymphocytes and monocytes/macrophage activity to microorganism or their antigen [36,37]. However, depleted T lymphocytes are associated with leishmaniasis [38,39]. So, we presume that in lymphocyte depleted condition, the elevated serum ADA might be contributed by monocytes during visceral leishmaniasis. To test our hypothesis, we stimulated monocytes with Leishmania donovani in vitro and measured the secreted ADA level in culture supernatant. We found a significantly higher ADA activity in leishmania stimulated culture supernatant compared to unstimulated (Fig 1, p<0.001, S1 Table). Hence, this result suggests that monocytes also contribute to the secretion of ADA during Leishmania donovani infection. However, during leishmania infection, neutrophils are the first cells to reach the site of infection [40]. Therefore, to test whether granulocytes contribute to the production of ADA or not, we measured the intracellular ADA activity in mononuclear cells and granulocyte lysate. We found a significantly increased ADA activity of mononuclear cell lysate in active VL, compared to active PKDL, treated VL and PKDL and healthy subjects (Fig 2A, p<0.05, S2 Table). However, we could not find any difference in ADA activity of mononuclear cell lysate between active and treated PKDL (Fig 2A, P>0.05, S2 Table). This is attributed to the difference in the systematic and local immune response. In previous study also it has been observed that the immune response of PKDL peripheral blood mononuclear cells varies from that of nodular/ macular cells [24]. Interestingly, we could not find any significant difference in granulocyte lysate ADA activity between VL, PKDL and healthy control before or after treatment (Fig 1,  p>0.05). Our intracellular granulocyte ADA activity results are in agreement with findings of Erel et al [25]. So, these findings indicate that mononuclear cells contribute to the elevated serum ADA activity during infectious diseases. Elevated serum ADA activities have been observed in many infectious diseases mainly involving the macrophages [22,23,41,42] and it has been considered as a marker of cell mediated immunity [43,44]. In our subsequent experiments, we measured the serum ADA level in VL and PKDL patients in their pre and post treatment stages. The level of serum ADA was found significantly higher in Indian patients with VL and PKDL at pre-treatment stage compared to healthy subjects (Fig 3A, p<0.05, S3 Table). After successful treatment the serum ADA level decreased to 3.0 and 2.3 fold in VL and PKDL patients respectively (Fig 3A,  p<0.05) and could not find any significant difference between VL and PKDL after treatment (Fig 3A, p<0.05, S3 Table). Interestingly, we found a significantly increased ADA level in active VL compared to active PKDL patients (Fig 3A, p<0.05). This discrepancy may be due to difference in immuno-pathogenesis of both forms of disease, VL is more aggressive and systemic disease, whereas PKDL is comparatively mild and restricted to the skin lesions [28,45,46]. Further, a linear correlation was observed between treatment course and serum ADA level in PKDL patients, serum ADA level subsides as the course of the treatment proceeds (Fig 3B,  p<0.05, S3 Table).Due to single dose Ambisome drug regime, the ADA activity could not be measured during mid treatment in VL cases. Interestingly, 2 out of 32VL subjects (K-442/14 and K-483/14) showed elevated serum ADA activity during follow up and within a week, they are admitted as relapse cases. These results indicate the correlation between the serum ADA level and onset of the disease, which emphasises its prognostic value.
Asymptomatic individuals have been shown to harbour the parasite in their blood [47] and these cases can act as potential reservoirs in transmission dynamics of leishmaniasis. Multiple lines of evidences revealed that, a different degree of asymptomatic individuals (5%-30%) convert to VL clinical manifestation [47][48]. The intriguing thing, why particular asymptomatic individuals will develop VL clinical manifestation and rest will self cured has not been solved yet [49]. Relapse cases in our study gave a clue that elevated serum ADA might be a cue for the onset of the disease. Therefore, we presumed that all the asymptomatic might not possesses similar serum ADA activity. To test this hypothesis, we evaluated the serum ADA activity of endemic healthy subjects and rK39 positive asymptomatic subjects. The serum ADA activity of endemic healthy subjects found to be par with normal range, but, asymptomatic subjects exhibit uneven serum ADA activities. Only 17.6% (six out of thirty four) of the endemic asymptomatic subjects showed elevated serum ADA activities (Fig 4, S4 Table). This result notions the plausibility that, these individuals may harbour the parasite in their body. Interestingly, previous reports suggest that, the immune response of asymptomatic individuals to leishmanial antigen is similar to cured VL patients [24]. Although, asymptomatic individuals and cured VL patients shown the similar immune response but there was a great variability in the response of asymptomatic subject to leishmanial antigen [50]. This variation might be attributed to the plausibility that majority of the asymptomatic individuals might behave like cured patients and rest might behave like VL patients. Similarly, in our study marginal number (17.6%) of the asymptomatic individuals showed elevated serum ADA activity which is similar to active VL or PKDL cases and rest showed normal serum ADA activity which is similar to healthy or treated patients (Fig 4).This evidence points to the conjecture that marginal number of the asymptomatic subjects may show the cell mediated immune response which results in the elevated ADA level in the serum. In summary, our analysis shows that, mononuclear cells secretes ADA in response to leishmanial infection and the elevated serum ADA activity is associated with VL and PKDL, and serum ADA activity can be used as prognostic marker to monitor the course of the treatment.

Conclusion
Visceral leishmaniasis (VL) constitutes a serious public health problem in endemic regions. Failure of early diagnosis jeopardizes the patient of an unfavourable evolution of the disease. In this context, an accurate diagnostic and an unambiguous prognostic marker will be a valuable tool for effective clinical practice. But, existing methods of leishmaniasis diagnosis and prognosis are hampered by the inherent limitations. On the other hand, ADA assay could be used as an alternative method of screening individuals with suspected VL and PKDL cases or as a tool for monitoring the efficacy of treatment and the appearance of relapse of subclinical disease. In our opinion, it is prudent to use rK39 strip and ADA in tandem, because seropositivty for rK39 and elevated serum ADA always associated with clinical leishmaniasis. Most importantly, this test is easy to perform, doesn't required expertise and cold storage, therefore this test is well suited for field condition. However, we feel a longitudinal study on this is needed for further evaluation of this test.
Supporting Information S1