Research:

Leishmania:

Sudan is considered as one of the most important areas of leishmaniasis in the world where sharp epidemics involving thousands of people with many deaths were recorded. All forms of leishmaniasis i.e. CL, MCL, VL and PKDL occur in Sudan. This group of diseases causes serious economic loss in the country, both in terms of the disability of affected individuals and in the cost of treatment, especially as most of those with leishmaniasis are on low incomes and live in rural areas.

Visceral leishmaniasis (VL) is the one of the most important endemic diseases in the country and is known to occur in the Sudan since 1904 when Neave (1904) described the first patient in the country. The main endemic area is in the eastern part of the 21  country, from the banks of the White Nile in the West to the Ethiopian border in the East, and from Kassala in the North towards Malakal in the South see Fig.(1). Other smaller foci have also been described in Kapoeta in Equatoria and parts of Kordofan and Darfur provinces. Occasional severe outbreaks occur, like the one in the southern Fung in Blue Nile province in 1956-1960, which caused thousands of death (Sati,1958). An outbreak of kala-azar was reported in Khartoum among displaced people (de Beer et al, 1990). Also, epidemics have occurred in recent years in war zones of southern Sudan where about 100,000 people died of leishmaniasis since 1984 (Seaman et al, 1996). Recently Roberts et al. (2000) reported that more than 10% of the population in southern Sudan died from visceral leishmaniasis over the past 5 years. Zeese and Frank (1987) estimated that of the total number of 1300 patients reported annually in the Sudan, more than 75% were treated in the hospitals of Gedaref and Hawata, a small rural town ~100km south to Gedaref, situated along the Rahad River. L. donovani senso lato is incriminated as the aetiological agent of VL (Hoogstraal & heyneman, 1969; El- Hassan et al., 1995). The incubation period of kala-azar appears to be between 2 months to 2 years (Zijlstra et al., 1991). P. orientalis is the only known proven vector of kala-azar in the Sudan (Hoogstraal & Heyneman, 1969; Elnaiem et al., 1997). However, in the Kapoeta area in South Sudan, where Porientalis is not known to be present P. martini may be the main vector of VL (Miniteret al, 1962). The Nile Rat (Arvicanthis niloticus) is being incriminated as the reservoir host for VL in Sudan (Hoogstraal & Heyneman, 1969; El- Hassan et al., 1995).

Figure 1: Endemic areas of visceral and cutaneous leishmaniasis in Sudan.

: Study area

\\\\\ : Cutaneous leishmaniasis

#### : Visceral leishmaniasis

Also Hoogstraal & Heyneman, (1969) reported the rodent Acomys aligena, The Spiny Mouse and two species of carnivores: Genetta g. senegalensis, The Senegal Genet and Felis serval phillipsi, The Sudanese Serval Cat to be infected.

The diagnosis of kala-azar is classically made on clinical grounds as well as parasitologically by examination of smears of lymph node, bone marrow or splenic aspirates. Lymph node aspirate has been recommended as a safe procedure with sensitivity of 78% in Sudanese kala-azar (Siddig et al., 1989). However, other serological tests were applied such as the enzyme- linked immunosorbent assay (ELISA) and the direct agglutination test -DAT- (Harith et al., 1986; El-Safi & Evans, 1989). Recently the PCR has been introduced for the diagnosis of leishmaniasis (Osman et al., 1997).

Kirk & Sati (1947) introduced the treatment of kala-azar with sodium stibogluconate in the Sudan and antimony is well established as the drug of choice (Zijlstra et al., 1993).

The only drugs affordable in poor countries are the pentavalent antimonials. Drug costs vary between about $20 and $200 for treating an average patient.

Post kala-azar dermal leishmaniasis (PKDL) is a known complication of VL in Sudan, although cases without previous history of kala-azar were reported in Sudan (El-Hassan et al., 1992).

Cutaneous leishmaniasis (CL) is common in the North and is characterized by ulcers in the skin that usually self heal, leaving a scar. In recent years a major epidemic occurred in large parts of northern Sudan, causing thousands of cases (El-Safi & Peters, 1991).

CL is caused by L. major (Abdallah et al., 1973). P. papatasi was incriminated as avector for CL (Abdallah & Sherif, 1978). Arvicanthis niloticus is believed to be the reservoir of cutaneous leishmaniasis in the Khartoum area (El-Safi & Peters, 1991).

Mucocutaneous leishmaniasis (MCL) in the Sudan is a rare condition, less than 100 cases have been described. The condition may be caused by L. major or it may follow kala-azar, which is caused by L. donovani (Ghalib et al., 1992).

KALA-AZAR IN A HIGH TRANSMISSION FOCUS: An ethnic and geographic dimension

M.E.Ibrahim, B.Lmbson, A.O, Yusif, N.S, Difalla, D, A.ALnaiem, A.Ismail, H.Yousif,

H.W.Ghalib, E.A.G.Khalil, a, Kadaro, D.C.BARKER, and A.M, ELHASSAN

         In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (Kala-azar) for evidence of infection with Leishmania donovani  . Leishmania DNA was detected by the polymerase chain reaction

In the peripheral blood of active, former patients of visceral leishmaniasis and also from   Asymptomatic subjects .using the cloned antigen rk39, antibodies were detected in44.2% Of game wardens while Leishmanin skin test result positive in77% of our samples .it was Shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan more likely to develop visceral leishmaniasis, whether this is due to genetic factors or previous exposure to Leishmania parasite remains to be elucidated.   

GENETIC VARIATIONS WITHIN   LAISHMANIA DONOVANI PARASITES REVEALED BY MINISATALLITE DNA MARKERS AND RLFP ANALYSIS

IN AN ENDEMIC AREA IN EASTERN SUDAN

ME Ibrahim, AM El Hassan, Muzamil Mahdi Abdelhamid

Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan;

The study aimed to determine intraspecific variations between strains of Leishmania donovani the eastern of Sudan using molecular approach, RFLP analyzing of cytochrome oxidase. SAcp2&HASPB2 as interagenic microsatalitte DNA were targeted for the study

The result suggested that RFLP analysis using Ssp1is a simple and easy to perform for characterization of Leishmania species .the analysis of Ssp-2 locus especially for the flanking markers demonstrated their usefulness in the detection of intraspecific variation 

In L.donovani parasites, while concurrently appears to be promising methods for epidemiological and taxonomic explorations, being sensitive and rapid, and able to illuminate a considerable molecular diversity and delineating the genetic relationship

Between the different species   

Genetic susceptibility to visceral leishmaniasis in the Sudan: linkage and association with IL4 and IFNGR1

HS Mohamed1, 2, ME Ibrahim1, EN Miller2, CS Peacock2, EAG Khalil1, HJ Cordell2, JM Howson2, A. M. El Hassan1, REH Bereir1and JM Blackwell

1. Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan;

2. Cambridge Institute for Medical Research

 Longitudinal studies in Sudan show ethnic differences in incidence and clinical phenotypes associated with Leishmania donovani. Immunologically, bias in type 1 vs. type 2 cytokine responses is important. To determine whether polymorphisms at IL4/IL9 or IFNGR1 contribute to susceptibility, we examined 59 multicase families of visceral leishmaniasis (VL) with/without post Kala-azar dermal leishmaniasis (PKDL). Multipoint nonparametric analysis (Allegro) linked IL4/IL9 to VL per se (P ¼0.002). Transmission disequilibrium testing with robust variance estimates confirmed association in the presence of linkage between VL per se and IL4 (P ¼0.008) but not IL9. Stepwise logistic regression analysis showed both IL4RP2 and IL4RP1 markers contributed significantly to the association, suggesting a common disease-associated haplotype. In contrast, IFNGR1 was linked (P ¼0.031) and associated (P ¼0.007) to PKDL but not VL or VL per se. Hence, polymorphism in a type 2 cytokine gene influences underlying susceptibility to VL, whereas IFNGR1 is specifically related to susceptibility to PKDL.

SLC11A1 (formerly NRAMP1) and   susceptibility to visceral leishmaniasis in The Sudan

HS Mohamed, ME Ibrahim, EN Miller, JK White, HJ Cordell, JMM Howson, CSPeacock, EAG Khalil1, AM El Hassan1and JM Blackwell

Genetic susceptibility to visceral leishmaniasis (VL) is indicated by differences in incidence and clinical phenotypes between ethnic groups in Sudan. In mice, innate susceptibility to Leishmania donovani, the etiological agent of VL, is controlled by Slc11a1 (formerly Nramp1). We therefore examined polymorphisms at SLC11A1 in 59 multicase families of VL from the high-incidence Masalit tribe in Sudan. Multipoint nonparametric analysis in ALLEGRO shows a significant linkage across SLC11A1 (Zlr scores 2.38–2.55; 0.008pPp0.012; information content 0.88). The extended transmission disequilibrium test shows biased transmission of alleles at 5.... polymorphisms in the promoter (P ¼0.0145), exon 3 (P ¼0.0037) and intron 4 (P ¼0.0049), and haplotypes formed by them (P ¼0.0089), but not for 3.... polymorphisms at exon 15 or the 3.... UTR. Stepwise logistic regression analysis using a case/pseudo-control data set derived from

the 59 families was consistent with main effects contributed by the intron 4 469 þ 14G/C polymorphism. Although the two alleles for 469 þ 14G/C lie on haplotypes carrying different alleles for the functional promoter GTn polymorphism, the latter did not itself contribute separate main effects. Sequence analysis of 36 individuals failed to identify new putative functional polymorphisms in the coding region, intron 1, intron/exon boundaries, intron 4/exon 4a, or in the 3.... UTR. One novel promoter polymorphism (–86G/A) was located within a putative nuclear factor kappa B binding site that could be functional. Further work will determine whether additional polymorphisms occur upstream in the promoter, which could be in linkage disequilibrium with the intron 4 polymorphism. These studies contribute to knowledge of the role of SLC11A1 in infectious disease.

Divalent cation transport and susceptibility to infectious and autoimmune disease: continuation of the Ity/Lsh/Bcg/Nramp1/ Slc11a1 gene story

 Jenefer M. Blackwell, Susan Searle, Hiba Mohamed, Jacqueline K. White

Cambridge Institute for Medical Research,

Solute carrier family 11 member a1 (Slc11a1), formerly known as Nramp1/Ity /Lsh/Bcg, is a proton/divalent cation antiporter that regulates susceptibility to infectious and autoimmune disease. Here we review recent studies on (1) the role of Slc 11a1 in iron metabolism and iron recycling in macrophages; (2) the use of mouse breeding and introgression of knockouts onto Slc11a1 congenic backgrounds for genes encoding the multiple pleiotropic functions associated with Slc11a1; and (3) associations/linkages of SLC11A1 with human disease and how these relate to functional promoter region polymorphisms.

A major susceptibility locus for visceral leishmaniasis maps to chromosome 6q27

 E. Nancy Miller, Manal Fadl, Christopher S. Peacock, Michaela Fakiola, Hiba S.Mohamed,  Abeer El Zein, Sarra E. Jamieson, Selma Jeronimo, Mary E. Wilson, Ashley Bales, Eltahir A. Khalil, Ahmed Elhassan, Ahmed M. Musa, Fernando Silveira, Jeffrey J.Shaw, Muntaser E. Ibrahim, Jenefer M. Blackwell

Major foci of visceral leishmaniasis (VL) caused by Leishmania donovani and L. chagasi occur in Sudan and Brazil, respectively. Familial clustering and ethnic differences suggest genetic control. A genome-wide linkage study was performed on two villages, El-Rugab and Um-Salala, located ~40 kilometers apart in eastern Sudan and occupied by the Masalit ethnic group who migrated from western Sudan in the 1980s. A 10 cM scan of 400 markers in 48 families (122 affecteds) provided evidence (LOD score 2.7; p=2.1x10 -4 ) for linkage to a 20 cM interval on chromosome 6q27. Refined mapping indicated two peaks within this interval. Addition of 21 families (51 affecteds) and stratification by village demonstrated a major gene at D6S281 (LOD score 3.07; p=8.6x10 -5 ) in El-Rugab only. Both villages contributed to the second peak at D6S396 (LOD score 1.35; p=006) on 6q26-q27. A broad region of linkage on chromosome 1 also resolved into two clear peaks upon stratification by village: at D1S2766 on 1p22 (LOD score 1.18; P=0.009) for Um-Salala and at D1S238 on 1q31 for El-Rugab (LOD score 1.25; p=0.008). Neither village provided evidence to support a VL susceptibility gene on 22q12 reported in the related Aringa ethnic group, also immigrants from western Sudan who settled ~100 kilometers from our villages in eastern Sudan in the 1940s. These data demonstrate the potential role that founder effect and population substructure may have in determining which genes act to regulate disease in different populations. Genome scan data from 132 nuclear families (215 affecteds) from ethnically admixed Brazilians provided supportive evidence for a gene at D6S281 on 6q27 (LOD score 0.63; p=0.044), and evidence for a population-specific

Role of Cytokines Gene Polymorphisms in Susceptibility to PKDL in Sudanese population

The leishmaniasis research group (LRG) in Sudan has been carrying out a longitudinal study in one of the highest endemic areas of Kala-azar in eastern Sudan for >10 years.  The incidence rate of VL and Post Kala-azar Dermal (PKDL), and the severity of the disease, were found to differ between different ethnic groups inhabiting the same study area (Masalit and Hawsa).  Our previous study was carried out to identify the role of a selected candidate genes, including SLC11A1 (formerly NRAMP1) encoding human natural resistant associated macrophage protein 1, IL4 encoding interleukin-4 and IFNGR1 in host susceptibility to VL.  The results showed significant linkage between polymorphisms at SLC11A1 and susceptibility to PKDL and VL (Mohamed et al., 2003b).  Interestingly, while polymorphism at IL4 contributed to underlying susceptibility to VL, variation at IFNGR1 appeared to be linked and associated specifically with susceptibility PKDL in the Masalit (Mohamed et al., 2003a).  Given the pivotal role of IFNg and IL10 in leishmanial infections, it would be worth pursuing the role of different polymorphisms at IFNGR1 and IL10 in susceptibility to PKDL in the susceptible Masalit population in The Sudan. So, that opens a gate of our study.

Ongoing research:

1- Role of IFNgR1 and IFNg polymorphisms  in susceptibility to PKDL in selected Sudanese Population (Mohmed Ahmed Mohmed Salih).

2- Role of IL10 polymorphisms  in susceptibility to PKDL in selected Sudanese Population (Shimaa Farouq A/rahman).

3- Characterization and genotyping of leishmaniasis among two villages in Eastern Sudan (Muna  Zumrawi)