Introduction

From a worldwide public health point of view zoonotic visceral leishmaniasis(ZVL) is one of the most important parasitic disease emerging in recent years( WHO,1990).Visceral leishmaniasis(VL) or kala azar is an endemic disease in parts of Iran,in some areas of Fars province in south and in Ardabil and East Azerbaijan in north-west(Edrissian GH ,et al 1988).In East-Azerbaijan province a high number of cases of kala azar were recorded during the last decade,which indicates that VL is endemic in some areas of this province.The study was carried out in Azarshahr during 2000-2001 showed that, by direct agglutination test(DAT) ,1.9% of the collected specimens (1252 blood specimens) were anti-leishmania antibody positive with ≥1:3200 titers and 1.2% with 1:1600 titres were suspicious( Misamadi N,et al 2003).This study was carried out during 2009-2010 in the villages of Azarshahr area of East-Azerbaijan provience.The aim was to determine the seroprevalence of VL in the area.

Material and Methods

The study area is situated in the south-east of Uromieh sea and have 50 Km distance from Tabriz city,at an altitude 1367 meteres.The investigation was carried out over a period of 12 months from September 2009 to October 2010 on the residents of 15 villages and 15 area of Azarshahr city.The study was desciptive (cross-sectional) and plasma samples were collected in heparinized capillary tubes from 10% of children ≤10 years old.Altogether,1500 blood specimens were collected to detect anti-leishmania antibodies.These samples were tested in the protozoology laboratory, faculty of medicine,Tabriz university of medical sciences by direct agglutination test(DAT) according to the methods described by Harith etal that prepared in the Protozoology Unit of the faculty of medicine in Tabriz university of medical sciences(Harith A etal,1988;Harith A etal,1989).The principal phases of the procedure for making DAT antigen were mass production of promastigotes of leishmania in the RPMI 1640+fetal bovine serum,trypsination of the parasites,staining with coomassie blue and fixing with formaldehyde.For making local DAT antigen,Leishmania donovani strain I-S kindly provided by Dr.AE Harith,that exist in protozoology unit ,Faculty of Medicine in Tabriz university of medical sciences,was maintained in the NNN medium enriched with LIT liquid phase (Ssdigursky M,Brodeskeym CT,1986) and used in our laboratory.

Results

Of the 1500 plasma samples prepared from children 19 cases (1.3%) showed anti-leishmania antibodies in titres of 1:3200 by DAT (Table 1).All the cases were children under 10 years.The highest proportion of positive cases was in in children <1 years old and the rate among children decreased with age. A number of seropositive cases (8) were found among children with no previous history of kala azar.About 1.3% of the seropositive individuals were males and 1.2% females,giving a male to female ratio of 1.02:1.

The geometric mean reciprocal titre (GMRT) in females (15848.9) was much higher than in males(5214.8).The patients were diagnosed clinicaly with kala azar.All of them were under 9 years old and 8(80%) were under 4 years.All of them were seropositive with DAT analysis ( titres ≥1:3200).Fever (36% of cases ) and splenomegaly (42.1%) were the most common signs and symptoms.

Figure 1

Table 1: Results of direct agglutination tests(DAT) from 1500 plasma samples by age in Azarshahr area,northwest of Iran

Figure 2

Table 2: Results of direct agglutination tests from 1500 plasma samples bay sex

Discussion

In this study, serological surveys using DAT analysis showed 1.9 % of the population in the study areas had anti –Leishmania antibodies in titres of >1:3200, all of them children under 10 years old . A number of seropositlve cases were found among children with no previous history of kala azar. The peak number of cases was in children < 1 years old and the seropositive rate decreased with increasing of age of the children. Prior studies in the Islamic Republic of Iran have shown a seropositive rate of about 50% in the age group 1-2 years and 96% of seropositive cases in children up to 8 years old [1-3].

About 1.3% of seropositive individuals were males and 1.2% females. No statistically different (P< 0.05) was observed between them.

Cross- sectional IFA and DAT serological survey of VL in endemic foci of the Islamic Republic of Iran showed that females are exposed and became infected at least as much as males.

However subclinical form of the disease may b more common in femals than males.

In some rural areas the rate of active kala azar cases in males may be higher than females [8].

Therefore, is dose not seem that VL affects males more than females, at least in the Azarshahr areas studied. However, the GMRT in females (15848.9) was very much higher the in males (5214.8).

Out of 19 diagnosed cases of kala azar four here, 68.4% were children less than 5 years old and all of them were under 10 years. All of them were seropositive (> 1: 3200 ) by DAT.

Fever and splenomegaly were the predominant Clinical features. These signs and symptoms are same as those found in other clinical studies [2].