Seroprevalence of Leishmania infection among the healthy blood donors in kala-azar endemic areas of Iran

Abstract

Visceral leishmaniasis (VL) is a serious health problem in Iran. The disease is mainly transmitted by sand fly bites, but its transmission through transfusion in endemic areas may also occur. The current study aimed to determine the prevalence of Leishmania infection among blood donors in VL-endemic areas in south of Iran. A total of 2003 healthy blood donors from blood service centers in five VL-endemic districts in Fars province, southern Iran, were enrolled in the study. The blood samples were assessed for antibodies against Leishmania infantum by direct agglutination test (DAT). Seropositive subjects were tested for the presence of L. infantum DNA in their buffy coat by the molecular method. Socio-demographic features of the subjects were also documented during sample collecting. The mean age of participants was 36.3 (SD = 10.7 years). Male constituted 94.7 % of the subjects while only 5.3 % of donors were female. Twenty-eight blood donors (1.4 %) were positive for Leishmania infection by DAT. Only one of these seropositive donors was positive for Leishmania infection by polymerase chain reaction. A significant correlation was found between age, the place of residence and seropositivity to Leishmania (P < 0.05). Findings of this study revealed that the prevalence of Leishmania infection among blood donors in transfusion centers in the VL-endemic areas in Iran is relatively high. These asymptomatic blood donors may constitute a risk of transmitting of VL to susceptible recipients.

Introduction

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania donovani complex including Leishmania infantum in Middle East and Mediterranean area and L. donovani in the Indian subcontinent and Eastern Africa (Desjeux 2004).

Cutaneous and VL are present in most of (14 of the 22) countries of the Eastern Mediterranean Region of the World Health Organization (Postigo 2010). Iran is an endemic country for both cutaneous and VL (Motazedian et al. 2002; Davami et al. 2010; Mohebali et al. 2011). VL is a major health problem in northwest and southern of the country. Dogs are the main reservoirs of the diseases, although feline leishmaniasis has also been reported from the region and cats may also be involved in the epidemiology of the disease (Hatam et al. 2010; Sarkari et al. 2009). The main foci of the disease in southern Iran are Kazeroun, Nourabad, Firouzabad and Darab districts in Fars province (Fakhar et al. 2008; Sarkari et al. 2010, 2012; Mohebali et al. 2011). Based on a hospital records, 260 cases of VL have been recorded during 2001–2009 from these regions (Sarkari et al. 2012).

Transmission of Leishmania through transfusion has been reported from different VL-endemic areas of the world (Cohen et al. 1991; Mauny et al. 1993; Cummins et al. 1995; Singh et al.1996). Many of frequent recipients of transfusion are patients undergoing treatment for malignancy, recipients of transplants, infants, and patients on steroid. These populations are at risk of acquiring VL through transfusion due to their immunosuppression status (Cardo 2006).

Blood donors in VL-endemic areas are healthy individual who may have Leishmania infection without having an apparent sign or symptoms. These asymptomatic infected subjects may transmit leishmaniasis to susceptible recipients through transfusion (Cardo 2006).

Studies carried out on healthy blood donors in VL-endemic areas have revealed different rate of Leishmania infection in these asymptomatic donors. In Southern France, L. infantum has been reported in 76 of 565 asymptomatic blood donors and cultures of blood have been positive in 9 donors (Fichoux et al. 1999). Rate of Leishmania infection in asymptomatic blood donors in an endemic area of Spain was reported to be 2.4 % by ELISA and 7.6 % by Western blot (Riera et al. 2004). Another study in Spain by Riera et al. reported a seropositivity rate of 3.1 % for Leishmania infection in blood donor (Riera et al. 2008). A low prevalence (0.3 %) of L. donovani among blood donors has been reported in a VL-endemic area in Bangladesh (Huda et al. 2013).

The current study aimed to evaluate the prevalence of Leishmania infection among the healthy blood donors in a VL-endemic area in Iran.

Materials and methods

Study population and sample collecting

Blood samples were taken from 2003 healthy blood donors from blood service centers in five VL-endemic districts in Fars province. The centers were in Kazeroun, Jahrom, Darab, Firouzabad and Nourabad districts. Socio-demographic features of the subjects were also documented during sample collecting. Ethical approval of the study was given by the ethics committee of Shiraz University of Medical Sciences.

Serological test

Sera were obtained from the fresh whole blood of the blood donors. Moreover, buffy coat was obtained from each blood sample for subsequent DNA extraction. Sera and buffy coats were kept at −20 °C until use. Sera samples were tested for anti-Leishmania antibodies by direct agglutination test (DAT). The DAT antigen was prepared in our serology laboratory in the department of parasitology and Mycology at Shiraz University of Medical Sciences. Mass cultivated L. infantum (Lon-49, Iranian strain) was used for preparation of DAT antigen. DAT was performed as previously described (Sarkari et al. 2010). Briefly, samples were diluted in serum diluent (physiological saline) containing 0.78 % β-mercaptoethanol and 0.2 % gelatin. Two-fold dilution series of the sera were made in a V-shaped microtitre plate, starting at a dilution of 1:50 and going up to a serum dilution of 1:6,400. DAT antigen (50 μl) was added to each well containing 50 μl of diluted serum. After 2 min of gentle shaking on a flat surface, the plate was covered with a lid and checked after overnight incubation at room temperature. An antibody titer of 1:800 and above was considered positive.

DNA extraction and PCR

DNA was extracted from the buffy coat of the samples. DNA of each sample was extracted using proteinase K and lysis buffer followed by phenol/chloroform/isoamyl extraction. Absolute ethanol was used to precipitate the DNA. Precipitated DNA was re-suspended in 100 μl of double distilled water and stored at 4 °C until use. Polymerase chain reaction (PCR) was performed as described by Rahbarian et al. (2009). The primers which were used are: LeishF (5′-CAA CAC GCG GCC TCC TCT CT-3′) AND LeishR (5′-AAA AGG TTG TCN GGG 3′). The thermal cycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles including denaturation at 94 °C for 35 s, annealing at 60 °C for 35 s, extension at 72 °C for 45 s, and final extension at 72 °C for 5 min. PCR products were separated by electrophoresis in 1.5 % agarose gel stained with ethidium bromide.

Analysis of data

The findings were analyzed by SPSS software (version 17), with a P value <0.05 considered as statistically significant. Chi squared and Fisher exact tests were used to compare the prevalence values relative to the characteristics of the donors.

Results

The study population included 2003 blood donors, consisting of 1896 (94.7 %) male and 107 (5.3 %) female, with age ranging from 17 to 66 years. The mean age of participants was 36.3 (±10.7) years. Most of the subjects were aged 25–35 years. Participants were blood donors from five blood transfusion centers in five different districts (all known as VL-endemic areas) in Fars province. From the total of 2003 blood donors which were recruited, 28 blood donors (1.4 %) were seropositive for Leishmania infection by DAT. Out of 28 positive samples, only one of the donors was positive for Leishmania infection by PCR. A significant correlation was found between the age of participants and seropositivity to Leishmania (P < 0.05).

Considering the residence of the blood donors, Firouzabad had the highest rate of infection (3 %), followed by Nourabad (1.8 %), Kazeroun (1.7 %), and Jahrom (0.5 %) districts. The differences in Leishmania seropositivity and residence of the participants were statistically significant (P < 0.05). The highest rate of infection (1.9 %) was found in the age group of 46–55 years while the lowest seropositivity was seen in the age lower than 25 years. The differences between age group and Leishmania infection were statistically significant (P < 0.05).

While most of the donors (71.9 %) were given blood on a regular basis, or had the history of blood donation, 28.1 % of them had no experience of blood donation before. No correlation was found between Leishmania seropositivity and ABO blood group (P > 0.05). Moreover, no association was found between Leishmania seropositivity and the occupations of participants (P > 0.05). Table 1 shows the socio-demographic characteristics of blood donors and relative seropositivity to Leishmania in this study.

Table 1.

Socio-demographic characteristics of blood donors and relative seropositivity to Leishmania in Fars province, southern Iran

Characteristics	Frequency (no.)	Percent (%)	Positive for anti-Leishmania antibodies		P value
			No.	%
Gender
Male	1,896	94.7	27	1.4	>0.05
Female	107	5.3	1	0.9
Age group
≤25	49	2.9	0	0	<0.05
26–35	632	31.6	7	1.1
36–45	561	28	8	1.4
46–55	463	23.1	9	1.9
56 Through higher	272	13.6	4	1.5
Residence
Kazeroun	403	20.1	7	1.7	<0.05
Jahrom	395	19.7	2	0.5
Darab	402	20.1	0	0.0
Firouzabad	405	20.2	12	3
Nourabad	398	19.9	7	1.8
Educational level
Uneducated	51	2.6	1	2	>0.05
Primary and secondary level	363	18.2	4	1.1
Post-secondary level	442	22.2	8	1.8
High school diploma	685	34.3	9	1.4
University level	480	24.1	6	1.2
Occupation
Employee	548	27.4	8	1.5	>0.05
Business	979	48.9	12	1.2
Housewives	100	5	1	1
Student	158	7.9	1	0.6
Laborer	88	4.4	3	3.4
Farmer and stockbreeder	94	4.7	1	2.9
Unemployed	35	1.7	13	17.8
Blood group
A	549	27.5	8	1.5	>0.05
B	489	24.5	7	1.4
AB	131	6.6	2	1.5
O	823	41.3	11	1.3

Discussion

The current study is the first report of Leishmania infection among healthy blood donors in south of Iran which aimed to assess the rate of Leishmania infection in blood donors and also to evaluate the presence of Leishmania in buffy coat of seropositive subjects. Samples have been taken from healthy volunteer blood donating people living in VL-endemic areas of Fars province, south of Iran.

Findings of this study revealed that more than one percent of healthy blood donors in VL-endemic areas of Iran are infected with Leishmania. So, the blood recipients, especially those with immunosuppressed status and infants, in VL-endemic areas of Iran might be at risk of acquiring VL through transfusion.

The ability of an organism to survive in the stored blood contributes to its transmission through transfusion. Recent studies carried out on blood donors from different VL-endemic areas of the world have revealed a relatively high rate of Leishmania infection in healthy blood donors (Luz et al. 1997; Fichoux et al. 1999; Riera et al. 2004; Colomba et al. 2005; Cardo 2006; Riera et al. 2008; Scarlata et al. 2008).

More than 90 percent of L. infantum and also L. donovani infection do not progress to clinically apparent diseases, in spite of the persistence of visceral infection (Engwerda et al. 2004). As a result, in any given VL-endemic area, there are a vast majority of infected people; among them are blood donors, which would be remained undetected.

It has been shown that Leishmania can circulate in asymptomatic blood donors more than 1 year after exposure (Fichoux et al. 1999; Cardo 2006). Therefore there is a risk of transmission of Leishmania to blood recipients due to its subclinical persistence in infected individuals.

Cases of transfusion transmitted leishmaniasis have been reported from different VL-endemic areas of the world including France, Belgium, Brazil and India (Cohen et al. 1991; Mauny et al. 1993; Cummins et al. 1995; Singh et al.1996). The majority of these infected recipients were patients who received whole blood from donors from the area where VL is endemic.

Support for the view of transmission of Leishmania through transfusion also comes from the study which has confirmed the transmission of Leishmania through blood transfusion in experimentally infected dogs (de Freitas et al. 2006).

Transmission of Leishmania through transfusion requires parasites to be present in the blood, either within infected white blood cells, or as free amastigotes released from phagocytes (Cardo 2006). Furthermore, the parasite should survive during processing and storage of blood. Disruption of monocytes during leukodepletion, or blood fractionation may result in release of amastigotes and could represent a potential transmission risk.

To avoid transfusional transmission of leishmaniasis, deferral of donation for as long as 12 months after returning from VL-endemic and permanent deferral of donation of people with apparent skin infection is taking place in the United States (Cardo 2006).

In our study, presence of antibodies against Leishmania was used for determining the prevalence rate of Leishmania infection in healthy blood donors. Nevertheless, antibody to Leishmania might not be detectable in all of asymptomatic subjects; therefore it may underestimate the prevalence of the Leishmania infection in healthy blood donors. In view of that, the real prevalence of Leishmania infection in blood donors might be higher than what we are reporting. In keeping with that, a study in Spain evaluated the blood donors for Leishmania infection by different methods and demonstrated the infection in 16/656 by ELISA, 50/565 by western blotting and 27/122 by PCR (Riera et al. 2004).

Previous studies demonstrated that Leishmania DNA may be present in the blood of healthy people without having any detectable humoral immune responses. In Reiera et al. study, only 2 of 18 PCR-positive donors were seropositive for VL. The lack of humoral immune response in asymptomatic individuals has been reported in Fakhar et al. (2008) study in Iran as well, where the individuals with positive PCR for Leishmania were seronegative by serological assays.

Considering the results of the aforementioned studies, some of the seronegative individuals in our study may have Leishmania infection.

In this study, the proportion of seropositive individuals increased with the increasing of the age of the subjects. This might be contributed to a boosting effect in asymptomatic infected subjects upon repeated exposure. Similar observation has been reported in Hasker et al. (2013) study.

Taken together, findings of this study revealed that healthy blood donors in VL-endemic areas of Iran can be infected with Leishmania. Therefore, the possibility of transfusion transmitted leishmaniasis in VL- endemic areas of Iran cannot be ruled out. Further studies are needed to find out the real risk of transfusion transmitted leishmaniasis in VL-endemic areas in Iran.