[Establishment and integration of a visceral leishmaniasis surveillance system in the primary health care system and its evaluation, Meshkin-Shahr District, Iran]

Visceral leishmaniasis is a systemic parasitic disease with a high fatality rate in under-5-year-old children. The disease is endemic in some parts of Iran, particularly in the north-west region. In 2001 a visceral leishmaniasis [VL] surveillance system was established for children aged>/=12 years in the primary health system in Meshkin-Shahr District, Ardebil Province, situated in the north-west of Islamic Republic of Iran. All cases with clinical signs and symptoms of VL and confirmed positive by the direct agglutination test [DAT] were referred for physical examination and treatment. The mean annual incidence of VL decreased significantly from 1.88 per 1000 children before [1985-2000], to 0.77 per 1000 child population after [2001-07], the intervention. In the control area with no surveillance, it increased from 0.11 to 0.23 per 1000. Early detection of VL using serological tests and timely treatment of cases can decrease the mortality and morbidity rates of VL in endemic areas

Sequence analysis of different domains of plasmodium vivax apical membrane antigen [PvAMA-1 gene] locus in Iran

Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 [AMA-1] is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far. P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid [AA] residues [42-488 of PvAMA-1] was amplified by PCR and cloned in pUC19 vector for sequencing. Sequence analysis of the antigen showed a high degree of identity [99%] with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea [Asian isolates] respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador. We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries

Integrated visceral leishmaniasis surveillance system in primary care for children in Meshkin-Shahr district, north-western Islamic Republic of Iran

In 2001 a visceral leishmaniasis [VL] surveillance system was set up for children aged

parasitological and serological study in malaria suspected patients in Hormozgan Province, Southeastern Iran

Presence of malaria immune factors induced by erythrocytic stages is widely used as an epidemiological approach to diagnose the infection mainly to distinguish the current, recent and past infections. This study was performed to find out the status of malaria, using microscopical and serological [IFA] methods in Bandar-Abbas and Minab, two malarious districts in Hormozgan Province of Iran. 408 patients with suspected malaria symptoms were enrolled. Conventional microscopic examination and serological IFA test were employed for diagnosis of malaria. The rates of agreement between microscopical and serological diagnosis were analyzed by Kappa test. 17.9% and 1.7% of the samples were microscopically diagnosed as P. vivax and P. falciparum, respectively. On the other hand, the serum samples were sero-positive with P. vivax and P. falciparum antigens in 54.2% and 32.1% of the samples, respectively. Serological IFA method could mainly determine the past history of malaria infection, but it was not helpful in detection of current infections. Moreover, there was no significant agreement between microscopical and serological [IFA] method0s in diagnosis of malaria

Evaluation of miltefosine against Leishmania major [MRHO/IR/75/ER]: In vitro and vivo studies

Cutaneous leishmaniasis is endemic in 88 different countries. There are an estimated 1.5 million new cases each year, with over 90% occurring in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria [Old World] and in Brazil and Peru [New World]. Miltefosine is effective in vitro and in vivo against Leishmania species and it was demonstrated efficacy in animals via the oral route. This study is the first one for evaluating the effect of miltefosine on cutaneous leishmaniasis of L. major [MRHO/IR/75/ER] by in vivo and in vitro studies in the BALB/c mouse model. As it was shown, miltefosine has a better effect on reduction of size of lesion compared to Glucantime[R], also it was not significant by statistical analysis. The results of this study show that miltefosine has a good activity against the proliferation of amastigotes of L. major. The results suggest that oral miltefosine might be a promising approach for developing new anti-Leishmanial drugs. [c] 2008 Tehran University of Medical Sciences. All rights reserved

[Seroepidemjological survey of visceral leishmaniasis by direct agglutination test in bojnoord district, north Khorasan province in 2007]

This study aimed to determine of the seroprevalence of visceral leishmaniasis to design a prevention and control program in Bojnoord district. This was a descriptive and cross sectional study with multistage random sampling method. The study was carried out on children up to 12 years old and 10% of adult's population in 8 rural areas of Bojnoord district in 2007. In total, 1608 blood samples were collected to detect anti-Leishmania antibodies. All the samples were tested by direct agglutination test [DAT]. In order to determine Leishmania species, necropsy was performed on four suspected dogs in the studied areas. The species of Leishmania was determined by RAPD-PCR and PCR-RFLP methods using to amplify the ribosomal internal transcribed spacer 1 [ITS1]. Thirty and eight [2.36%] out of 1 608 collected blood samples had anti- Leishmania antibodies at titer 1:800 and nine [0.56%] were just positive at 1:3200 by DAT. There was no statistically significant difference between female and male seroprevalence [p>0/05]. The seoprevalence in children <=12 years old compared to individuals greater than 12 years old did not show any statistically significant [p>0/05]. Amastigotes were observed in all 4 necropsied dogs. The species of Leishmania isolated from 2 dogs, was determined as L.Infantum. Their ITS1 sequences were registered with Accession numbers EU810776 and EU810777 in NCBI. These findings showed that visceral leishmaniasis has been circulated with low endemicity in Bojnoord district. Therefore an appropriate monitoring system is needed for health services in this area

Seroepidemiological study on canine visceral leishmaniasis in Meshkin-Shahr district, Ardabil province, northwest of Iran during 2006-2007

This study aimed to determine the seroprevalence of canine visceral leishmaniasis in Meshkin-Shahr district as endemic areas of human visceral leishmaniasis [HVL] for presenting control program of HVL to health authorities. A Seroepidemiological study to determine seroprevalence of canine visceral leishmaniasis [CVL] among ownership dogs using direct agglutination tests [DAT] in 21 villages of Meshkin-Shahr district, Ardabil Province was carried out from June 2006 to August 2007. Three hundred and eighty four ownership dogs were selected by multi-stage cluster sampling. Chi-square and Fisher exact tests were used to compare seroprevalence values relative to gender, age and clinical signs. Of the 384 serum samples tested by DAT, 17.4% [95%C.I, 13.2%-20.8%] were positive [1:320 and higher]. No statistical significant difference was found between male [16.5%] and female [20.2%] seroprevalence [P=0.416]. The highest seroprevalence rate [64.2%] was observed among the ownership dogs of three years age and above. Only 25.4% of the seropositive dogs had clinical signs and symptoms. The most clinical signs among symptomatic dogs were cachexia [75%] and alopecia [36.5%]. The majority of seropositive dogs [74.6%] lived in endemic areas of Meshkin-Shahr district were asymptomatic. It seems that all symptomatic and asymptomatic infected dogs are the most important risk factors for human infection in VL endemic areas

Leishmania tropica: another etiological agent of canine visceral leishmaniasis in Iran

Dogs have been previously reported to be reservoirs of Leishmania infantum as the etiological agent of human visceral leishmaniasis in Iran. We report a case of canine visceral leishmaniasis [VL] caused by L. tropica from the north- west of Iran where human visceral leishmaniasis is endemic. The canine VL was initially screened by dipstick rK39 and direct agglutination test, then the dog was dissected and obtained samples were examined by parasitological [direct exam, cultivation] and molecular techniques [RAPD-PCR and RFLP-PCR]. Leishmania parasites were found in spleen and liver of the dog. The serological tests for the detection of specific anti-leishmania antibodies showed positive results. L. tropica as another agent of canine VL was determined

Malaria in Iran: past and present situation

Malaria had being widely prevalent for a long time in Iran. Before starting any anti-malarial campaign in Iran about 60% of population was living in malaria endemic areas. In hyper-endemic areas, approximately 30 to 40% of the total mortality was due to malaria. The malariometric data, reported during 1921-1949 in the malaria surveys in some endemic areas, showed high endemicities of the disease in most parts of the country. The first malaria-training course for preliminary operations of anti-malaria campaign was started in Iran in 1945. Afterwards, in the courses conducted, mostly by the Institute of Malariology many technical personnel were trained. In 1947, for the first time DDT was used in mosquito control in a pilot study in malaria hyper-endemic villages near Tehran. It caused great reduction in malaria transmission. Anti-malarial campaign including drug prophylaxis and treatment, anti-mosquito spraying with DDT and some anti-larval control measures, carried out during 1948-1956, considerably decreased malaria infection rate in most endemic areas. In 1957, malaria eradication programme [MEP] started in Iran and up to 1980 almost interrupted malaria transmission in the north parts of the country. However, in the south parts although the infection rate considerably decreased, but due to some technical and operational problems, malaria transmission was not interrupted. Therefore, in 1980 the MEP shifted to malaria control programme [MCP] which has been continuing up to present time. From 25 species of Anopheles found in Iran, 8 species of A. stephensi, A. fluviatilis, A. culicifacies, A. pulcherimus, A. d_thali, A. superpictus, A. sacharovi and A. maculipennis are considered to be malaria vectors. The prevalent species of Plasmodia in Iran are P. falciparum and P. vivax. P. malariae is rare. The main problems, in the malaria endemic areas of the southeast parts of Iran are resistance of the main vectors to some insecticides as well as high resistance of P. falciparum to chloroquine. The total reported malaria cases in Iran from 96340 with 45% P. falciparum in 1991, gradually, decreased to 18966 with 12% P. falciparum in 2005. About 30 to 50% of malaria patients have been among foreign immigrants

Application of direct agglutination test [DAT] for the diagnosis and seroepidemiological studies of visceral leishmaniasis in Iran

Visceral leishmaniasis [VL] is one of the most important parasitic diseases which is endemic in different parts of Iran. Serological studies were conducted by direct agglutination test [DAT] on 12144 human serum samples, collected from four geographical zones of Iran. Sero prevalence, geographical distribution, clinical signs and symptoms for human visceral leishmaniasis based on DAT for the period of 2002 through 2005 were determined. From 516 kala-azar cases detected: 50.6% were from Meshkin-shahr and Moghan districts in Ardabil Province, northwest of Iran and 49.4% were detected from other areas of Iran. In physical examination of seropositive cases, which were detected by DAT with anti-leishmanial antibodies at titers of 1: 3200 to 1: 102400, almost 50% of suspected individuals showed the classical kala-azar signs and symptoms. Predominant signs and symptoms in 233 hospitalized patients with anti-Leishmania antibodies at 1:3200 and higher, were fever [88.0%] and splenomegaly [84.5%]. Statistically significant difference was found between males [58%] and females [42%] [P< 0.01]. Moreover, 93.6% of the VL patients were < 5 yr of age, and 6.4% were older than 5 yr that this difference was statistically significant [P< 0.01]. From 1383 serum samples collected from domestic dogs in the villages that are known as endemic foci of human leishmaniasis, 152 [11.0%] were positive by DAT [>/= 1:320]. Parasitological and serological examinations that were performed in 30 wild canines showed that 10% of these animals were infected by L. infantum. L. infantum Lon49 is the principal agent of the disease in human as well as animal reservoir hosts in different parts of Iran. For the first time in Iran, L. tropica isolated from both skin lesions in the face and bone marrow aspiration in a HIV+ man who co-infected with VL as well as in an infected dog from Ardabil Province

Application and evaluation of PCR in detection of malaria in donors of transfusion centers in Sistan-Baloochestan province in 2002

After hepatitis and AIDS, malaria is the most prevalent transfusion outcome in endemic areas. Presence of asymptomatic carriess of malaria parasites in the endemic areas can be a source of infection in transmission of malaria by blood transfusion. Prevention of malaria caused by blood transfusion depends on screening blood donors and deleting infected blood samples. To screen blood samples, parasitological, serologic and molecular methods have been applied. In this study 120 blood donors in Iranshahr in Sistan-Baloochestan province were tested with different methods of thick and thin blood films, Immuno-Fluorescent Antibody Test [IFAT], and Polymerase Chain Reaction [PCR]. The result of all thick and thin blood films were negative. IFAT by using P.vivax antigen and P.falciparum antigen for 38 and 6 donors respectively showed a titre of antibody equal to +/- 1/20-1/320 [17 of the former group and 4 of the latter had a history of malaria infection]. The PCR assay using silica for DNA extraction and using P .falciparum specified primers with sensitivity rate equal to 2-3 parasites per microlitre of blood was negative for all subjects under study. This study showed, although microscopic examination of blood smears was inexpensive and simple, but it is labor-intensive and time-consuming that makes it insensitive for detection of low-level parasitemia in asymptomatic donors and for screening a large number of specimen. IFAT would not always show the real existence of parasites and in spite of simplicity and sensitivity because of its disability to be automated is not suitable for screening a large number of specimen. On the other hand, IF AT in individuals with malaria history and absence of parasites in their blood may be positive for a long period. It was approved that molecular methods such as PCR were more sensitive and more specific than conventional microscopic examination and their great advantage was the ability to detect the infection with low-level parasitemia that may have been distinguished by blood films examination. In the present study, probably because of low number of specimen or limited study duration with PCR method, or probably since parasitemia exiting in the subjects under study was less than 2-3 parasites per microlitre of blood, we were not able to detect positive cases

Effects of anti-mosquito salivary glands and deglycosylated midgut antibodies of anapheles stephensi on fecundity and longevity

With the aim of controlling malaria by reducing vector population, the effects of antibodies produced against salivary glands and deglycosylated midgut antigens of Anopheles stephensi mosquitoes on fecundity and longevity of the same species were tested. Three deglycosylated preparations of midgut and two preparations of salivary glands were produced, conjugated with aluminum hydroxide gel, and subcutaneously injected to shoulders of TO [Turner Out-bred] mice. After 4 immunizations and assurance of enough antibody production against utilized antigenic suspensions, effects of blood feeding on immunized and control mice were assayed. Insoluble preparation of midgut showed the strongest effect with 23.5% reduction in egg laying, and increasing death rate of vectors in third day after feeding. No significant reduction in fecundity or survivorship was seen with other preparations. Anopheles midgut insoluble antigens are potential candidates for designing vaccines against malaria vectors and further investigations need to be done to find effective antigens and the best way of their use

Identification of leishmania sepsis isolated from human cutaneous leishmaniasis, using r and om amplified polymorphic DNA [RAPD-PCR]

This cross-sectional study was designed to isolate of Leishmania spp from cutaneous leishmaniasis patients and characterized them by RAPD-PCR technique. Eighty- seven Leishmania isolates from 112 samples were collected from cutaneous leishmaniasis [CL] patients who referred to Mashhad Health Centers from August 2002 to May 2004. Desirable samples [87 isolates] were characterized by RAPD-PCR method using four selected oligoprimers. Electrophoresis patterns from each isolate were compared with reference strains of L. major, L. tropica and L. infantum. The results showed that 94.2% and 5.8% of isolates were similar to L.tropica and L.major reference strain, respectively. Four isolates that were determined by RAPD-PCR as L.major, could produce ulcer at the base tail of BALB/c mice, 4 - 12 weeks after inoculation but none of L. tropica isolates produced any lesions at the site of injection in the animals. The results indicate that L. tropica species are dominant in the studied areas of Mashhad city and RAPD-PCR technique is a suitable tool for Leishmania characterization in epidemiological studies

In-vivo monitoring of the response of falciparum and vivax Plasmodia to chloroquine in Bandar-Abbas and Kahnoudj, South-East Iran, 1997-1999

The in-vivo 28 days extended test was applied for monitoring of the responses of P. falciparum and P. vivax to chloroquine in malaria patients referred to Malaria Research Laboratory in Bandar-Abbas Training and Health Research Center. The selected patients were treated with standard dose of chloroquine [25 mg/kg over 3 days]. Primaquine was also administered in a single dose [0.75 mg/kg in the third day] as gametocytocidal in falciparum and weekly [0.75 mg/kg/w for 8 weeks] as anti-relapse in vivax cases. From 76 falciparum malaria patients, 63 cases were followed up for 28 days; in 40 patients [63.5%] the parasites were resistant to chioroquine at RI and RII levels. In 13 patients that the asexual forms of P. falciparum were disappeared in their blood by the day 7 and they were not accessible more than one and two weeks, the response of the parasite was considered as either sensitive [S] or resistant at RI level [SRI]. The rate of the chioroquine-resistant cases of P. falciparum among Afghan refugees was higher [86.3%] than the rate in Iranian patients [51.2%]. Totally, there is no significantly difference of chloroquine-resistant rate and levels of P. falciparum in the studied areas in compare to the results of previous study. In 323 vivax malaria tested patients the mean of parasite clearance time [MPCT] was 2.91 days [ranged 1-5 days] and the parasite is still highly sensitive to chioroquine

Seroepidemiological study of visceral leishmaniasis among humans and animal reservoirs in Bushehr province, Islamic Republic of Iran

Using direct agglutination tests, a survey of visceral leishmaniasis was carried out among children and adults from 13 villages and from nomadic tribes in Bushehr province during 1998-99. Of the 1496 plasma samples, the overall seropositive rate [titres > or = 1:3200] was 3.4%. Almost all cases [94.1%] were in children under 10 years old. Eighteen patients were diagnosed with kala azar; fever and splenomegaly were the predominant signs and symptoms. Parasitology and serology examinations of local animals identified dogs and jackals infected with Leishmania infantum. Suggestions for control of visceral leishmaniasis in this area are to eliminate stray dogs, identify cases among humans and suspected leashed dogs, and treat infected individuals

Application and evaluation of direct agglutination test in sero-diagnosis of visceral leishmaniasis in man and canine reservoirs in Iran

Direct agglutination test [DAT] was compared with indirect fluorescent antibody test [IFAT] for the diagnosis of visceral leishmaniosis [VL] in patients suspected of kala-azar and in dogs. Parasitological examination was also performed in some cases. A total of 2,295 plasma samples, collected from suspected kala-azar patients, were tested by DAT. Sero-positive rate [SPR] in titers of%1:3200 was 39.2%. The SPRs in 1,951 samples tested by both DAT and IFAT were 39.7% and 45.0%, respectively. The coincidence of the two tests was 85.0%. In 750 samples tested with the two DAT antigens. The coincidence of total positive and negative results was 93.6%. In parasitological examination of 207 cases suspected of kala-azar, amastigotes were found in 20 cases [9.6%]. However, SPR with DAT was 42.5%. In 11,174 total blood samples collected on filter paper from children [%10 yrs.] mostly in the endemic areas of Meshkin-Shahr, SPR with DAT was 14.9% and 74 children found to be new sero-positive cases. Serological surveys for VL in 303 dogs in the endemic areas of Ardebil province SPR was 14.9% for DAT and 20.0% for IFAT. In most of sero-positive dogs amastigotes were also found in parasitological examination

Kala-azar in Iran

From 1949 when the first case of Kala-azar was reported in Iran, to the end of 1988, at least 1361 diagnosed cases of this disease have been reported or recorded from all 24 provinces of the country. Visceral leishmaniasis is present in endemic form in the two provinces of Fars and East- Azerbaijan. In other provinces sporadic cases of Kala-azar have been found so far. The disease has been found mostly among children up to eight years of age: 96.5% in endemic focus of E.Azerbaijan and 80.8% in the sporadic cases, with a significant difference. The peak percentages of the number of the patients were 50% in the age group of 1-2 years in the endemic focus and 30% in the age group of 3-4 years in the sporadic cases. The percentages of the males among the Kala- azar patients of the endemic focus and sporadic cases were 59.1% and 64.1% respectively, with no statistically significant difference. It has been found in the endemic focus of East- Azerbaijan province, that females are affected as much as males. Domestic and stray dogs as well as wild carnivores such as jackals and foxes have been found infected viscerally with Leishmania. It seems that dogs are the main sources of infection for human visceral leishmaniasis in Iran, particularly in the endemic focus of E.Azerbaijan. The species of the sandfly vector has not been determined yet. Phlebotomus major is considered to be the main vector in most parts of the country. Ph.kandelakii can also be a suspected vector of visceral leishmaniasis in the endemic focus of East Azerbaijan province

Malarial antibodies and glucose-6-phosphate dehydrogenase [G-6-PD] deficiency

The IFA and the blue dye decolourization G6PD tests were applied in three cross-sectional studies to find out were applied in three cross-sectional studies to find out the relationship of malarial antibodies and G6PD deficiency in the male residents of the malarious areas of southern Iran. In the first study that the blood samples were collected in a random sampling method from the whole Hormozgan province, the G6PD deficient individuals has, significantly, lower sero-positive rate [SPR] and also, considerably lower total geometric mean of reciprocal titers [GMRT] with P.falciparum antigen as compared to the G6PD normal subjects. But with P.vivax antigen SPR and GMRT in both groups were almost the same. However in the second and third studies that the blood samples were collected from the selected groups of the residents of the above Hormozgan province with high incidences of malaria no such distinct serological differences between G6PD deficient and G6PD normal groups was observed. It is concluded that the cross-sectional serological survey of malaria in populations who are more frequently exposed to malarial infections, particularly in the areas where P.vivax is also prevalent is not enough to show a conclusive serological evidence to support G6PD /malaria hypotheses