Evaluation of Urine as a Diagnostic Specimen for Visceral Leishmaniasis in Sudan

The diagnosis of visceral leishmaniasis (VL) can either be direct via the demonstration by culture of microscopy of Leishmania parasites in lymph node, bone marrow or spleen aspirates or indirectly through serology or molecular biology [1]. Direct demonstration is invasive and requires technical expertise and a certain high level of medical precautions, in particular in the case of splenic aspiration (often considered to be the reference standard), as this has the risk of serious bleeding. Molecular biology is not yet field applicable and therefore serological tests, like the direct agglutination test (DAT) or rK39 immunochromatographic rapid tests are often employed as diagnostic methods [1]. However, serology still requires collection of blood samples which has an inherent risk of accidents, is a taboo in some countries and resented by some groups such as small children. Therefore, research into alternative diagnostic methods is not only looking for better applicable techniques, but also for patient samples that can be obtained in a non-invasive manner, such as urine.


Introduction
The diagnosis of visceral leishmaniasis (VL) can either be direct via the demonstration by culture of microscopy of Leishmania parasites in lymph node, bone marrow or spleen aspirates or indirectly through serology or molecular biology [1]. Direct demonstration is invasive and requires technical expertise and a certain high level of medical precautions, in particular in the case of splenic aspiration (often considered to be the reference standard), as this has the risk of serious bleeding. Molecular biology is not yet field applicable and therefore serological tests, like the direct agglutination test (DAT) or rK39 immunochromatographic rapid tests are often employed as diagnostic methods [1]. However, serology still requires collection of blood samples which has an inherent risk of accidents, is a taboo in some countries and resented by some groups such as small children. Therefore, research into alternative diagnostic methods is not only looking for better applicable techniques, but also for patient samples that can be obtained in a non-invasive manner, such as urine.
There are several reports, in particular from the Indian subcontinent that describes a good diagnostic performance of rK39 strip tests using urine samples [2][3][4]. In contrast, limited information on testing of urine samples with serology is available from East Africa (Sudan and Ethiopia), another VL hot spot, although it is well known that both tests have an overall good sensitivity and specificity in that particular region when performed on serum samples; DAT: 94.23% and 89.97%, respectively and rK39: 94.48% and 88.75%, respectively [5].
Therefore, we initiated a preliminary evaluation of rk39 strip tests and DAT on urine samples collected in Sudan, which is reported here.

Materials and Methods
Urine samples were collected from individuals in the catchment area of Umkoraa Rural Hospital in West of Gedarif State and Al Azaza Damus Kala-azar Clinic in Sennar State, which is an endemic area for malaria with seasonal transmission, and one of the most important VL areas in the world. Patients were diagnosed at recruitment in Sudan for malaria based on clinical symptoms and light microscopy of a thin blood smear, and were included in this study when the blood smear was positive for asexual parasites, identified as P. falciparum. VL diagnosis was based on clinical symptoms (including irregular fever, weight loss, splenomegaly and hepatomegaly) and serological testing; unfortunately parasitological testing was not performed. VL cases were included when a positive DAT on serum was obtained, with a titre>1:1600, or when DAT titre was 1:1600 and rK39 Kalazar Detect Rapid Test (InBios, International Inc., USA) or IT-Leish (Bio-Rad, France) strip test on serum was positive. The presence of a malaria infection in these cases was excluded through microscopy on a blood smear. This resulted in a total of 26 VL patients, 18 malaria patients and 17 patients with malaria-VL co-infections to be included in the study. The samples were kept on ice during transportation, and were stored without preservative at -20°C at KIT, Amsterdam, Netherlands.
In addition, 3 samples from apparently endemic healthy controls (EHCs) from Sudan and 5 urine samples from non-endemic healthy controls (NEHC), in the Netherlands, were included in the study. In the laboratory, urine samples were tested with the rK 39 Kalazar Detect Rapid Test, using an adapted protocol [2]. In brief, 150 μl of urine was applied directly to the test strip, without chasing buffer, and results were examined after 10 min. A test was considered positive when both the control line and the test line were visible, and negative when only the control line was visible. If the control line was not visible, a test was considered invalid.
The protocol of the FD-DAT, manufactured by KIT (Amsterdam, the Netherlands), was adapted for the use of urine. A 100 μl whole urine sample was added to the first row of a 96-well plate, and was two-fold serially diluted with 50 μl of saline+0.1M β-mercaptoethanol. 50 μl of reconstituted freeze-dried Leishmania antigen was added to each well, and results were examined the next day. Each plate contained a positive or negative control. Results were interpreted as negative when only a blue dot was visible and positive when agglutination was observed, with a cut-off value of 1:8 (based on prior testing with urine samples Abstract Diagnostic methods for visceral leishmaniasis (VL) require invasive specimen sampling. Urine is a potential noninvasive alternative and in the present study the diagnostic performance of direct agglutination test (DAT), based on a freeze dried antigen, and rK39 strip test (InBios, Bio-Rad) on specimens collected in Sudan was assessed. RK39 test had a sensitivity of 72.1% and a specificity of 76.9% on urine and DAT sensitivity was 62.8% and its specificity 69.2%, using initial diagnosis (VL diagnosis was confirmed on clinical and serological basis) as reference in both cases. Tests agreements were fair. Both rK39 as well DAT have potential in diagnosing VL using urine, but results are currently not as good as on the Indian sub-continent. from NEHC). The agreement between both diagnostic test and initial clinical diagnosis or between DAT and rK39 on urine in general was determined by calculating Kappa (k) values with 95% confidence intervals using Epi-info version 6 [6].

Results and Discussion
The results of testing urine samples with rK39 strip test or DAT for VL are presented in table 1. The rk39 test had a sensitivity of 72.1% (95% CI: 57.3-83.3) and a specificity of 76.9% (95% CI: 58.9-88.9) using the initial diagnosis (clinical symptoms combined with positive serology) as reference. The sensitivity of DAT on urine was 62.8% (95% CI: 47.9-75.6) and specificity 69.2% (50.0-83.5).These sensitivities and specificities are lower than those observed on the Indian sub-continent [2][3][4], but are better than those of another urine based diagnostic test, KAtex, in East Africa [7]. The difference between Indian and East African level of specificity and sensitivity could be explained by the heterogeneity of L. donavani strains in East Africa versus their homogeneity in India [7].
It is noted that the agreement between the general performance of DAT and rK39 on urine samples is good ( Table 2). The present study has some limitations. First, the total number of cases studies is low, but the work is intended as a pilot study to assess whether there is some potential for urine testing for VL diagnosis. A follow-up study will include more cases and all case definition must be improved, in particular with respect to the VL cases. The urine samples have been stored without a preservative and transported prior to analysis and this might have affected tests' performance [3]. Interestingly, rK39 and DAT performed almost equally on urine samples in the present study. This is in contrast to studies using serum samples from East Africa in which the rK39 test is reported to have a lower sensitivity and specificity [7]. In particular it is noted that several urine samples of malaria cases were also found positive, which cannot be explained at this stage, although it might be possible that these patients have an underlying asymptomatic leishmaniasis infection, as was demonstrated in India [8], which could contribute to this positive reaction with urine.
In conclusion, the present study has demonstrated some potential of diagnostic testing of urine with DAT and rk39 test for VL, but efforts to further improve test performance should be undertaken. Furthermore, it would be of interest to assess if, and how many (percentage) urine samples stay positive after a complete cure (as a possible test of cure).

Author's contributions
EvR conducted diagnostic tests in the Netherlands and drafted the manuscript; BYMN collected field samples, performed initial diagnostic analysis in Sudan and commented on the manuscript; HDFHS designed the study, supervised diagnostic testing in the Netherlands and finalized the manuscript. In section B the agreement between the different test/diagnosis is presented. The agreement between the tests was determined by calculating Kappa values with 95% confidence intervals using Epi-info version 6. Kappa values express the agreement beyond chance 6 .