Investigating the dynamics of <i>Leishmania </i>antigen in the urine of patients with visceral leishmaniasis: a pilot study

Prakash Ghosh; Israel Cruz; Albert Picado; Thomas Edwards; Md. Anik Ashfaq Khan; Faria Hossain; Rajashree Chowdhury; Emily R. Adams; Rupen Nath; Joseph M. Ndung'u; Dinesh Mondal

doi:10.12688/f1000research.16181.1

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Research Article

Investigating the dynamics of Leishmania antigen in the urine of patients with visceral leishmaniasis: a pilot study

[version 1; peer review: 2 approved with reservations, 1 not approved]

Prakash Ghosh^1,2, Israel Cruz³, Albert Picado³, [...] Thomas Edwards⁴, Md. Anik Ashfaq Khan², Faria Hossain², Rajashree Chowdhury², Emily R. Adams⁴, Rupen Nath², Joseph M. Ndung'u³, Dinesh Mondal²

Prakash Ghosh^1,2, Israel Cruz³, [...] Albert Picado³, Thomas Edwards⁴, Md. Anik Ashfaq Khan², Faria Hossain², Rajashree Chowdhury², Emily R. Adams⁴, Rupen Nath², Joseph M. Ndung'u³, Dinesh Mondal²

PUBLISHED 21 Sep 2018

Author details Author details

¹ Department of Formulations, IDRI, Seattle, WA, 98102, USA
² Nutrition and Clinical Services Division, icddr,b, Dhaka, 1212, Bangladesh
³ Foundation for Innovative New Diagnostics, Geneva, 1202, Swaziland
⁴ Research Centre of Drugs and Diagnostics, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK

Prakash Ghosh
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Israel Cruz
Roles: Conceptualization, Methodology, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Albert Picado
Roles: Conceptualization, Methodology, Project Administration, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Thomas Edwards
Roles: Investigation, Methodology, Resources, Writing – Review & Editing

Md. Anik Ashfaq Khan
Roles: Data Curation, Investigation, Methodology, Visualization

Faria Hossain
Roles: Data Curation, Investigation, Methodology, Visualization

Rajashree Chowdhury
Roles: Investigation, Methodology, Visualization, Writing – Original Draft Preparation

Emily R. Adams
Roles: Conceptualization, Funding Acquisition, Methodology, Project Administration, Resources, Writing – Review & Editing

Rupen Nath
Roles: Investigation, Methodology, Visualization, Writing – Original Draft Preparation

Joseph M. Ndung'u
Roles: Funding Acquisition, Project Administration, Resources, Writing – Review & Editing

Dinesh Mondal
Roles: Conceptualization, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Detection of Leishmania antigens in the urine provides a non-invasive means of diagnosis and treatment monitoring of cases of visceral leishmaniasis (VL). Leishmania antigen load in the urine may vary between different time-points within a day, thus influencing the performance of antigen-detection tests.
Methods: We investigated the dynamics of Leishmania antigen in urine collected at three different time points (08:00, 12:00 and 16:00 hours). All urine samples collected were tested with the Leishmania Antigen ELISA (VL ELISA) kit, produced by Kalon Biological Ltd., UK.
Results: The median concentration of Leishmania antigen in urine collected at 08:00 (2.7 UAU-urinary antigen units/ml) was higher than at 12:00 (1.7 UAU/ml) and at 16:00 (1.9 UAU/ml). These differences were found to be statistically significant (08:00 vs. 12:00, p=0.011; 08:00 vs. 16:00, p=0.041).
Conclusion: This pilot study indicates that the Leishmania antigen concentration is higher in urine samples collected in the morning, which has important implications when the VL ELISA kit or other tests to detect Leishmania antigen in urine are used for diagnosis of VL and treatment monitoring.

Keywords

Visceral Leishmaniasis; Leishmania Antigen; ELISA; Urine; Diagnosis; Treatment monitoring

Corresponding authors: Joseph M. Ndung'u, Dinesh Mondal

Competing interests: No competing interests were disclosed.

Grant information: This work was supported by funds from the Ministry of Foreign Affairs, Government of the Netherlands (Activity Ref. Nr. 22211, Developing Innovative Diagnostics to Address Poverty-related Diseases; www.minbuza.nl).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Ghosh P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Ghosh P, Cruz I, Picado A et al. Investigating the dynamics of Leishmania antigen in the urine of patients with visceral leishmaniasis: a pilot study [version 1; peer review: 2 approved with reservations, 1 not approved]. F1000Research 2018, 7:1514 (https://doi.org/10.12688/f1000research.16181.1) First published: 21 Sep 2018, 7:1514 (https://doi.org/10.12688/f1000research.16181.1) Latest published: 21 Sep 2018, 7:1514 (https://doi.org/10.12688/f1000research.16181.1)

Introduction

Visceral Leishmaniasis (VL), also known as kala-azar, is a potentially fatal vector-borne disease that, in the Indian subcontinent, is caused by Leishmania donovani protozoa, which are transmitted by female Phlebotomus argentipes sand flies¹. The number of cases of VL per year worldwide is estimated to be 0.2–0.4 million, with 20,000 to 40,000 associated deaths. Just six countries, in which the disease is transmitted as part of an anthroponotic (Bangladesh, India), zoonotic (Brazil) or a probable anthropozoonotic cycle (Ethiopia, South Sudan, Sudan), account for 90% of VL cases worldwide^2–4.

In patients presenting with VL-compatible signs, namely fever for more than two weeks plus splenomegaly and/or weight loss. VL is usually diagnosed by serology, either with a direct agglutination test or rK39 antigen-based rapid diagnostic tests. When parasite confirmation is required, the main approach is tissue aspirate microscopy (from spleen, bone marrow and to a lesser extent lymph node), which has a variable sensitivity and, because of the invasiveness of the procedure (especially spleen aspiration), requires experienced personnel and should be performed in hospitals where blood transfusion and surgical facilities are available. Besides, the accuracy of microscopic examination is influenced by the ability of the laboratory technician and the quality of the reagents and equipment used^4,5. Parasite confirmation by tissue aspirate microscopy is also used for treatment monitoring, test-of-cure (TOC) and diagnosis of relapses, since serology is useless for this purpose, as anti-Leishmania antibodies may remain detectable up to several years after cure^5,6. Initial cure rates vary between 49% and 94%. Therefore, alternative, less invasive options to invasive tissue aspiration and microscopy are needed to monitor treatment responsiveness, diagnose relapses and assess cure. Although molecular methods, such as PCR, have shown to be effective in VL diagnosis and treatment monitoring using less invasive samples; unfortunately, these require sophisticated laboratory and trained personnel, and there are no standardized protocols that can be used across endemic settings, which hinders their application^7,8.

Antigen detection tests, ideally in less invasive samples such as blood, serum/plasma or urine, are an interesting option, as antigen levels should reflect the parasite load in the patient. These tests also present an advantage over antibody detection in immunocompromised patients with low antibody response, as in Leishmania/HIV coinfection⁹. In chronic infections, such as VL, the detection of antigens of the pathogen in blood or serum/plasma can be complicated by the presence of high levels of antibodies, circulating immune complex, serum amyloid, rheumatoid factors, and autoantibodies, all of which may mask immunologically important antigenic determinants or competitively inhibit the binding of antibodies to free antigens¹⁰. Nevertheless, Gao et al.¹¹ proved that it was possible to detect Leishmania antigen in the sera of VL patients from China with high sensitivity and specificity. However, many of the problems described above may be avoided by searching for antigens in urine. Several studies have demonstrated Leishmania antigens in the urine of VL patients using different approaches, such as countercurrent immunoelectrophoresis, Western blot, latex agglutination test and ELISA^12–16.

Fluctuations in the quantity of Leishmania antigens excreted through urine might influence the sensitivity of these assays. According to the Clinical and Laboratory Standards Institute guidelines, and confirmed by other authors, urine collected in the early morning contains urinary components at the highest concentration and is more reliable for quantification of urine markers^17,18. However, there is no evidence concerning the persistence and levels of Leishmania antigen in urine collected in the early morning versus other time points. Therefore, given the utility of antigen detection tests in VL diagnosis and treatment monitoring, we set out to study the dynamics of Leishmania antigens in urine in order to determine which time point is the most appropriate to detect Leishmania antigens in VL patients using the Leishmania Antigen ELISA (VL ELISA) kit (Kalon Biological, Ltd., UK). Further, in a recent study we showed that the parasite load in relapse VL is higher than the primary VL cases¹⁹. Therefore, we hypothesized that the level of Leishmania antigens in urine might differ in different states of VL. In our current study we compared the Leishmania antigens level in patients with primary VL and relapse VL.

Methods

Study sites and subjects

This study was conducted at the Emerging Infections and Parasitology Laboratory, International Centre for Diarrheal Disease Research, Bangladesh (icddr,b), between 15 March and 30 April 2016. The study population was a convenience sample of 16 patients with VL (seven primary VL, seven relapse VL and two with treatment failure) who were invited to participate in the study while hospitalized at Surya Kanta Kala-azar Research Centre (SKKRC), the only specialized hospital for VL treatment in Bangladesh. Patients were eligible if they had VL. Patients in the study were grouped as type-1 (primary VL) or type-2 (patients presenting with either relapsed disease or treatment failure). The patients were diagnosed according to the national guidelines for VL management in Bangladesh: a patient from a VL-endemic area presenting with fever for more than 2 weeks, splenomegaly and positive by rK39 rapid diagnostic test (here, Kalazar Detect^TM, InBios Intl., USA was used). Information on clinical and demographic characteristics of the patients is provided in Table 1.

Table 1. Clinical and demographic characteristic of the VL patients included in the study (N=16).

Variable	Value
Age in years, mean ± SD	33 ± 21
Children (<17 years), n (%)	3/16 (19%)
Adults (≥17 years), n (%)	13/16 (81%)
Male, n (%)	8/16 (50%)
Splenomegaly, n (%)	16/16 (100%)
Fever for ≥2 weeks, n (%)	16/16 (100%)
Duration of fever in days, mean ± SD	20.25±4.20
ESR, mean ± SD*	88.3 ± 11.92
Hepatomegaly, n (%)	11/16 (69%)
Darkening of skin, n (%)	14/16 (87%)
Pancytopenia, n (%)	7/16 (43%)

*mm/h. ESR, erythrocyte sedimentation rate.

Specimen collection

Urine samples were collected at 4-hour intervals compatible with routine activities at SKKRC from each of the 16 enrolled patients before initiation of treatment. A total of 50 ml midstream urine was collected in a tube containing 0.1% NaN₃ at 8:00, 12:00 and 16:00 hours. Immediately after collection, all samples were stored at -20°C in SKKRC facilities and then transported to icddr,b, maintaining the cold chain. A 2-ml aliquot of urine from each of the subjects and time points was used for this study.

Leishmania antigen ELISA

The Leishmania Antigen ELISA (VL ELISA) (Kalon Biological Ltd., UK) uses a set of polyclonal antibodies against non-proteic Leishmania antigens. As the antigens detected in urine with this kit remain largely uncharacterized, the unit Urinary Antigen Unit (UAU) is used to express the amount of Leishmania antigens detected. ELISA was performed according to the manufacturer’s instructions, described elsewhere¹⁶. Briefly, samples were diluted using the assay diluent provided with the kit and a 1:20 dilution was used to determine the antigen concentration. A total of 100 µl diluted urine were tested in triplicate together with duplicates of the antigen calibrators included in the kit using 96-well ELISA plates. After incubation at room temperature optical density (OD) was read at 450 and 620 nm (Biotek, microplate reader). OD at 620 nm was subtracted from OD at 450 nm for further calculations of UAU. A four-parameter logistic standard curve was constructed for each plate using the calibrator provided with the kit. Then Leishmania antigen level in each sample was estimated from the standard curve.

Statistical analysis

The difference between antigen concentrations at three different time points of all urine samples was investigated. Based on the distribution of data, a non-parametric test (Wilcoxon matched-pairs signed Rank test) was performed to determine significant differences between medians. To find out any correlation between the antigen concentrations at different time points with participants’ age, Spearman’s test was performed. Mann-Whitney U-test was performed to investigate the difference in the antigen concentrations at different time points within sex and the difference between type-1 and type-2 patients. Statistical analyses were performed using the GraphPad Prism software version 7.03 and SPSS version 20.0.

Ethics approval and consent to participate

This study was approved by the icddr,b Ethical Review Committee, research protocol number PR-14093. Informed written consent was collected from each participant, or the legal guardian in the case of children.

Results

The median concentration of Leishmania antigens was 2.7 UAU/ml, 1.7 UAU/ml and 1.9 UAU/ml in urine samples collected at 08:00, 12:00 and 16:00, respectively (Figure 1). Most of the study subjects (9/16, 56.3%) showed highest urinary Leishmania antigen concentration at 08:00 (Table 2). The five patients presenting the highest antigen concentration at other time points had either identical or similar levels at 08:00. Only two patients (ID, 7 and 16) showed a marked decrease in antigen concentration at 08:00 compared to the 16:00. The median concentration of Leishmania antigens in urine collected at 08:00 was significantly higher than the median concentration of Leishmania antigen in urine collected at 12:00 (p=0.011) and at 16:00 (p=0.041) (Figure 1). However, we did not find significant differences in the Leishmania antigen levels between urine samples collected at 12:00 and 16:00 (p=0.820). Further, the investigation did not find any correlation between the antigen concentrations at different time points with participants’ age and sex (Table 3). In addition, the concentration of antigen in urine of primary VL cases did not differ with the antigen concentration in patients with VL relapse or treatment failure (Table 3).

Figure 1. Difference between median antigen concentrations (UAU/ml) in urine samples collected at three indicated time points.

Table 2. Description of participants and antigen concentrations at different time points.

Bold figures indicate highest daily concentration.

Patient ID	Patient VL type	Time of antigen concentration assessment
Patient ID	Patient VL type	08:00, UAU/ml	12:00, UAU/ml	16:00, UAU/ml
1	Relapse	0.757	0.488	0.857
2	Primary	1.016	0.885	0.602
3	Relapse	12.587	12.133	1.842
4	Treatment failure	3.103	3.2	2.031
5	Primary	6.415	5.923	1.301
6	Relapse	1.31	0.366	0.226
7	Primary	0.484	0.266	3.109
8	Treatment failure	37.292	10.146	7.897
9	Relapse	28.784	3.937	7.072
10	Primary	2.383	1.7	1.028
11	Primary	6.161	7.088	3.567
12	Primary	1.915	1.688	1.836
13	Relapse	52.269	23.290	52.4
14	Relapse	4.940	1.497	2.541
15	Relapse	0.678	0.916	0.786
16	Primary	0.422	0.477	2.664
Mean [UAUAg]		10.03	4.62	5.60
SD [UAU]		15.05	6.17	12.63
Median [UAU]		2.7	1.7	1.9
IQR		0.8-11.04	0.6-6.8	0.9-3.45

VL, visceral leishmaniasis; UAU, urinary antigen unit.

Table 3. Correlation between participants’ age, sex and disease type with the antigen concentrations in urine collected at three different time points.

Time points	Correlation between participant age and Ag concentration, r (P)	Difference in Ag concentration by sex		P value	Difference in Ag concentrations by Patient type		P value
Time points		Male median (IQR)	Female median (IQR)	P value	Type 1, median (IQR)	Type 2, median (IQR)	P value
8:00	-0.411 (0.114)	4.04 (1.1-24.73)	2.74 (0.53-6.05)	0.505	1.92 (0.48-6.2)	4.95 (1.03-33.04)	0.174
12:00	-0.441 (0.088)	2.81 (0.60-9.4)	1.6 (0.60-5.24)	0.721	1.7 (0.48-5.92)	3.2 (0.70-11.14)	0.408
16:00	-0.435 (0.092)	1.84 (0.70-6.2)	2.3 (1.1-3.0)	0.721	1.84 (1.03-3.11)	2.03 (0.82-7.5)	0.758

Type 1, Primary VL; Type 2, VL relapse or treatment failure.

Dataset 1.Details of patient symptoms, demographic information and results of ELISA for Leishmania antigens.

Leishmania antigen load is not found in this Dataset, but can be found in Table 2.

Discussion

One of the antigen detection tests most widely used in VL diagnosis is the KAtex latex agglutination test (Kalon Biological, Ltd., UK). Although the first studies showed very promising results, further evaluations proved that this test returns variable sensitivity (36–100%) and specificity (64–99%)²⁰, which has limited its wide use for both diagnosis and treatment.

Given the potential applications of Leishmania antigen detection tests, the interest in developing new approaches has been sustained and recent efforts have resulted in the development of two standardised, user-friendly, quantitative and direct ELISA tests that may prove to be useful for VL diagnosis and treatment monitoring; the Leishmania Antigen Detect^TM ELISA (InBios International Inc., USA) and the Leishmania Antigen ELISA (VL ELISA) (Kalon Biological, Ltd., UK). Moreover, strategies for VL control in the Indian subcontinent (ISC) seems to be working well, currently the main foci to be considered are South America and Eastern Africa, being VL zoonotic in the first and anthropo-zoonotic in the second. Till date, no study has yet been performed to evaluate this test for canine leishmaniasis where it might have the potential to diagnose canine leishmaniasis thereby improving surveillance of reservoirs, proper treatment, monitoring transmission and assessing the efficacy of control activities in endemic areas including South America and the Mediterranean basin where canine leishmaniasis is an important veterinary issue.

Although these two ELISAs have the potential to be useful for treatment-monitoring in human VL, they also showed that at the same time point, especially at the day of diagnosis, the parasite load can be very different from patient to patient¹⁶. This could be due to the fact that patients are not in the same moment of the VL episode when they seek for diagnosis, or because the samples were taken at different times of the day. In this pilot study we have tried to address the second explanation, and have found that the highest level of Leishmania antigen in urine is obtained with early-morning urine samples. A recent study explored that urine collected in the early morning improves the sensitivity of urinary lateral flow LAM assay for diagnosis of TB in HIV- infected patients, which is congruent with our study finding²¹.

The Kala-azar Elimination Programme in the ISC has been conducting diverse activities since 2005, with active case detection being one of the key activities to stop transmission of VL²². However, to eliminate the disease, proper follow-up of treated VL cases and prompt relapse management is no less important, since in the ISC 1–16% of treated VL patients relapse and 10–20% develop post kala-azar dermal leishmaniasis (PKDL)^16,23. At present icddr,b, in collaboration with the Liverpool School of Tropical Medicine and the Foundation for Innovative New Diagnostics, is evaluating the efficacy of the Leishmania Antigen ELISA (VL ELISA) kit for diagnosis of VL, PKDL and asymptomatic infection in Bangladesh. Thus it is critical to ensure that the urine samples are taken at a time and in conditions that increase the chances of detecting Leishmania antigens. In this pilot study we have assessed the dynamics of Leishmania antigens in urine from VL patients attending the SKKRC hospital in Bangladesh, and we have found that urine collected at 08:00 contains the highest amount of Leishmania antigens. These findings can be used as a guide to ensure the best performance of the Leishmania Antigen ELISA (VL ELISA) kit when used either for VL diagnosis or treatment monitoring, as well as for implementation of this method in endemic regions in future where this disease is zoonotic. Furthermore, prospective studies are warranted to explore the efficiency of Leishmania Antigen ELISA (VL ELISA) kit as a predictor of VL relapse.

Conclusion

The Leishmania antigen load in the urine of VL patients varies at different times during the day, and is highest in the morning. This should be taken into account in order to increase the sensitivity of the Leishmania Antigen ELISA (VL ELISA) kit, and to harmonize sample collection time points during treatment follow-up, so the comparison of the measurements taken on different days can be reliably compared.

Data availability

Dataset 1. Details of patient symptoms, demographic information and results of ELISA for Leishmania antigens. Leishmania antigen load is not found in this Dataset, but can be found in Table 2. https://doi.org/10.5256/f1000research.16181.d217632²⁴.

Grant information

This work was supported by funds from the Ministry of Foreign Affairs, Government of the Netherlands (Activity Ref. Nr. 22211, Developing Innovative Diagnostics to Address Poverty-related Diseases; http://www.minbuza.nl).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We are grateful to all the study participants. We are also thankful to icddr,b and its core donors: GOB, UKAID, USAID and SIDA.

Faculty Opinions recommended

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