Spectrum of Leishmania donovani infection in the Southwest of Sudan: A rapid epidemiological mapping

From the early 1900s, visceral leishmaniasis (VL) has been among the most important health problems in Sudan. The disease is historically endemic in a wide belt extending from the western bank of the White Nile and the Sudan–Ethiopian border. This area includes the southern areas of Central and Eastern states and the north-eastern part of Upper Nile State. Since 1989, VL has spread in the western part of Upper Nile State, the disease has never been reported before. Cases have also been reported from Darfur State in western Sudan, the Nuba mountains area (Kordofan State) and the Kapoeta area in Eastern Equatoria [1].

The advent of GIS has greatly facilitated the mapping process and, in particular, enables maps to be updated rapidly and presented in different ways. CL and VL could possibly be mapped using GIS and database of the incidence and/ or prevalence rates in a selected area using census data and offi cial CL/VL records from the available health centres or peripheral hospitals [2][3][4].
In this study we aimed to identify Leishmania spectrum of infection in an endemic area in Western Sudan by using rapid assessment method that included: Clinical history, leishmanin skin test, Direct Agglutination Test (DAT), as a rapid epidemiological assessment scheme which is very much needed to prevent unnecessary loss of lives.

Abstract
This epidemiological survey was based on simple immunological techniques (leishmanin skin reactivity and the direct agglutination tests), combined with clinical history to obtain data about the spectrum of L. donovani infection in communities at risk of developing visceral Leishmaniasis. The data was depicted in map format to give an enhanced visual impact.
In this study clinical history and immunological tests, together with the past history of the infection, were combined and conducted in 1781 randomly selected volunteers in Southwestern Sudan.

Ethical consideration
This study was ethically approved by the Ethical Boards

Sample size for seroepidemiological screening
We randomly recruited 1781 individuals; demographic and clinical data was collected in a specially designed questionnaire by direct interviewing and entered into the computer. Data from laboratory tests was also entered.

Volunteer's assessment
All selected volunteers were primarily subjected to clinical interview for their previous exposure to VL infections. They were then subjected to leishmanin skin testing (LST) as described by Zijlstra et al. The test was read after 48 or 72 hours using the ballpoint pen method [5]. The presence of induration +/-erythema of ≥5 was considered a positive reaction [6]. Direct agglutination test (DAT) was also performed as described by [7]. The test is a direct reaction between antigen (L.donovani whole parasite antigen) and antibodies (patients serum). Briefl y, patients' sera were diluted (1:100-1:102400) in gelatine diluents. 50μL of the antigen was added in each well of V-shaped microtiter plates. The test was read in the next day and a titer of ≥1:3200 was considered as a cut-off for a positive test.

Parasite isolation and cultivation
A lymph node aspiration was performed for all suspected VL patient who had lymphadenopathy. Those whose diagnosis were not confi rmed by direct microscopic examination but were still highly suspected were contacted in the next day and preceded for bone marrow aspiration. Aspirations from enlarged lymph node were done using a sterile needle, the epitrochlear lymph node was punctured and then the materials were injected into a vacutainers containing NNN culture medium. Prior to bone marrow aspiration, patients were fi rstly being locally anaesthetized, and then using a bone marrow aspiration needle, a few drops of the bone marrow were taken from the posterior superior spinae. The materials were also dispensed into containers of culture medium. Samples were kept at 26˚C before being transferred to the laboratory where it was properly kept at 27˚C for 2 weeks until promastigotes reached their stationary phase.

Map drawing
The study area coordinates were obtained using GPS 12 XS, Garmin International, Kansas, USA. ArcGIS Version 9.0, ESRI, USA was used to construct a project called VL/GIS project (VL/ GIS.apr) and fi les contained layers coverage's e.g. political boundaries; populated areas; drainage network, drainage points and roads network were downloaded. The interactive dynamic map was drawn and all coverage layers were placed in one view. Tables of leishmanin reactivity, DAT titres, age and sex distribution for the selected villages were added to the table and appropriate charts were also made in the project. Different layouts were prepared to show map themes and then the study area was constructed. The weather phenomena including total temperature, rainfall average, humidity, and wind directions were collected from National Metrological Centre, Khartoum, Sudan.

Results
The mean age of the volunteers was 20.7 years (range 1-90). Children (<15 years) who are the most susceptible group to VL formed 53% of the study volunteers. Past history of VL (treated at nearby hospitals) was reported in 11%. History of skin rash following VL treatment was not reported. The majority of the volunteers (75.5%) did not react to the leishmanin antigen, but those above 15 years of age were more likely to be leishmanin reactive (induration >5mm) compared to those below that age. DAT titres <3200 have been noticed in 88% of the volunteers, whereas 11.7 had a titers ≥3200. A comparative summary of all of these results is shown in Figure 1.
Moreover, 63 individuals had a history of parasitology confi rmed VL within the previous 12 months. Some of them presented and were diagnosed during the survey period. The mean age of these patients was 9.7+/-6.0 years, with an equal male: female ratio. All were non-reactive to leashmanin skin testing and their DAT titers were equal and/or above 3,200.

Mapping the results
The constructed project was named VL/GIS. The project contained views that were formed from political boundaries, drainage network and road network themes. Tables of age groups, leishmanin reactivity and DAT titres were added to the cases to the project. Charts were constructed from the tables.

Discussion
Visceral leishmaniasis (VL) is a major health problem in eastern and southern parts of the Sudan and it mainly affects children below 15 years Zijlstra et al., [8][9][10].
The western parts of the country are not known to be endemic with cutaneous leishmaniasis, but sporadic VL cases were detected. These areas witnessed a huge outbreak that has killed an estimate of 100.000 lives in late eighties. Such outbreak revealed the inadequacy of health services in dealing with such disasters that are more likely to occur from time to time [11].
The demographic data collected showed that the communities under study are mainly of the young age groups with approximate equal numbers of males and females.
The past history of VL infection was lower than the rate of leishmanin conversion; this may simply point to the possibility of subclinical infection [7,12]. Past history was considered as knowledge about VL in that area is fair enough as well as people's diagnosis about the disease is very good. Leishmanin skin test (LST) was chosen as a simple and cheap test that correlates well with development of cell-mediated immunity and exposure to leishmania as was reported previously from eastern Sudan [8][9][10]13]. In this study, the LST positivity increased in direct proportion with age indicating that as individuals get older they will be increasingly exposed; some will develop overt disease while others remain asymptomatic (subclinical infection). This was extensively reviewed Bucheton et al., [12].
Volunteers who had DAT titers equal and above 6,400 match closely those who gave past history of VL. As reported by [14,15], that positive DAT test results may be obtained from patients with active VL, subclinical infection, and those who were infected more than 7 years previously (DAT titre will remain high for many years following successful treatment). The fi ndings of this study are in agreement with that reported earlier by Hashim et al., [11] and recently by Mohammed et al., [16], who described an outbreak of VL among the Mesairya tribe in Kordofan area, this tribe moves annually towards Bentue areas looking after their cattle in search of water and suitable pasture, and they may acquired the infection during their stay in that areas. There is no transmission of VL in the Misairiya homelands around Muglad and Babnusa, but some of them may contract the infection during their daily movement and keeping close to the animals i.e. cow odours are very attractant to sand fl ies, which could easily be hidden in the animal hairs and then the fl ies activates and could easily transmit the parasites Baleela et al., [17].
Several authors use GIS technology for mapping leishmaniasis using spatial data and environmental factors [4,18]. Golpayegani et al., [3], investigated the relationship between environmental factors (vegetation and elevation) affecting the prevalence of cutaneous leishmaniasis and visceral leishmaniasis. After mapping the prevalence data  Moreover, High-case incidence rate (CIR) plays a major role in the spatial dispersion of the VL cases between non-endemic and endemic villages [4].
In this study the geographic information system (GIS) have been used as an early alarm system to determine those (Supplementry Tables)