Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Genotyping of Echinococcus granulosus isolated from canine in Northwest Iran.

Echinococcus granulosus is a parasitic tapeworm that causes cystic echinococcosis, a potentially life-threatening zoonotic infection affecting humans and animals across the globe. In Iran, the prevalence of this parasite remains a significant public health concern, particularly in the northwest region. This study aimed to investigate the genotypes of E. granulosus isolated from canines in the northwest of Iran. A total of 87 samples were collected from the Mughan plain area in Ardabil province, including 47 stray dogs (Canis familiaris), 25 golden jackals (Canis aureus), and 15 red foxes (Vulpes vulpes), and molecular analysis was performed for partial cytochrome c oxidase subunit 1 and nad1 genes. Phylogenetic analyses were carried out on the obtained sequence. The findings revealed that 9 out of 87 (10.3%) samples were infected with Echinococcus parasites, with a frequency of 1 (4%) and 8 (17%) among golden jackals and stray dogs, respectively. Overall, all (100%) E. granulosus adult samples were related to the G1 genotypes. This study provides comprehensive data regarding the epidemiological and molecular characteristics of echinococcosis in canines in northwest Iran.

Visceral Leishmaniasis in Iran: An Update on Epidemiological Features from 2013 to 2022.

Background: Visceral leishmaniasis (VL) is one of the most important neglected tropical diseases. The zoonotic form of VL is endemic in some areas of Iran. We aimed to determine the status of VL identified in humans and canines in different parts of Iran from 2013 to 2022. Method: A national representative cross-sectional study was conducted in 10 provinces of Iran, including the national leishmaniasis reference lab. We employed the direct agglutination test (DAT) as a reliable serological method to detect anti-Leishmania infantum antibodies in humans and animal reservoir hosts. Additionally, a narrative literature review was conducted to identify relevant studies on VL seroprevalence in Iran from 2013 to 2023. Results: The results of 21281 human and 5610 canine serum samples from 2013 to 2022 are reported. Altogether, 448 (2.1%, 95%CI: 2.0-2.3) human serum samples showed anti-L. infantum antibody levels of ≥1:3200. Of these samples, 13716 (64.5%) were collected actively, which showed a seroprevalence of 0.6% (95% CI: 0.5-0.8) and 7565 (35.5%) were collected passively, which showed a seroprevalence of 4.8% (95%CI: 4.3-5.3). Overall, 1035 (20.1%, 95%CI: 19.0-21.2) of 5160 domestic dogs (Canis familiaris) samples showed anti-L. infantum antibody levels of ≥1:320. Northwest (2.8%) and northeast (0.96%) regions had the highest human VL seroprevalence, while northwest (21.5%) and south (14.4%) regions had the highest canine VL seroprevalence. Conclusion: Zoonotic VL, an endemic parasitic disease, is still present in several different distinct areas across Iran. While human VL cases have shown a declining trend over the last decade, the prevalence of canine VL remains significant.

Molecular Identification of Cutaneous Leishmaniasis Species in the Northcentral Iranian Province of Alborz: Is There a New Focus on Cutaneous Leishmaniasis in the Province?

Background: Cutaneous leishmaniasis (CL) is an endemic infection in the Middle East, including Iran that is also spreading to new foci. We aimed to determine the leishmaniasis species causing CL in Alborz province. Methods: Overall, out of 55-suspected CL patients referred to health centers in Alborz Province, north central Iran in 2019, 40 patients had positive smear for CL based on optical microscopy. The internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA (rDNA) was amplified by PCR. Leishmania species were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) using BshF I (Hae III) enzyme. Results: Out of the 40 positive patients with CL, 34 cases (85%) had been caused by Leishmania (L) major and six (15%) by L. tropica. Fifteen patients had no history of traveling to the disease endemic areas, of which nine were Iranians. Skin lesions and scars caused by CL were mostly observed on the hands and face. Moreover, more than two skin lesions were observed in 22 cases (55%), all of which were infected with L. major. A single skin ulcer was seen in 18 (45%) of the CL patients. Conclusion: Climate change, reduced rainfall, and demographic changes such as migration into Alborz Province and the increasing marginalization of the population and their entry to settle in new areas might have caused natural transmission of both L. tropica and L. major in this province.

The Association of Human Leucocyte Antigen (HLA) Class I and II Genes with Cutaneous and Visceral Leishmaniasis in Iranian Patients: A Preliminary Case-Control Study.

Background: Leishmaniasis is currently considered a re-emerging or emerging infection based on the geographic region. The outcome of leishmaniasis vastly depends on Leishmania-host interaction. This preliminary study aimed to show the association of human leukocyte antigen (HLA) class I and II genes with healed and non-healed cutaneous leishmaniasis (CL), and symptomatic and asymptomatic visceral leishmaniasis (VL) compared with control groups in Iran. Methods: Ninety-five people, including 31 patients versus 64 individuals in the control group, were enrolled. Among them, 20 patients had confirmed CL based on amastigote observation, 10 had improved CL and 10 non-healed CL. Eleven patients were suffering from confirmed VL based on direct agglutination test (Five asymptomatic and six symptomatic VL cases). Besides, they were residents in an endemic area of VL in the northwest of Iran. To select a control group, it was ensured that they had no history of leishmaniasis. Peripheral blood samples were collected from each patient. After DNA extraction, HLA typing was conducted using polymerase chain reaction - sequence-specific priming (PCR-SSP). Subsequently, data were statistically analyzed by SPSS. Results: There was a statistical relationship between the presence of HLA-A26 and CL, healed CL and the existence of the B38 allele, C1 allele and symptomatic VL, as well as B1.4 allele and asymptomatic VL (P<0.05). Conclusion: This primary finding indicates that several HLA genes have a potential role in the susceptibility of Iranian people to CL and VL.

Seroprevalence of Visceral Leishmaniasis in Children Up To 12 Years Old of Rural Areas from Kermanshah Province, Western Part of Iran.

Background: After the earthquake in 2017 a few new cases of visceral leishmaniasis (VL) were reported from SarPol-e-Zahab district of Kermanshah Province, western part of Iran. This study was conducted to determine the seroprevalence in Kermanshah Province. Methods: This descriptive cross-sectional study was conducted on children up to 12 years of age from SarPol-e-Zahab County, Kermanshah Province, western part of Iran in 2021. For each individual, a questionnaire including age, sex, clinical features, history of the disease, and contact with canines as reservoir hosts of VL were completed, separately. To determine VL seroprevalence, blood samples were collected from the children and after centrifugation, the sera samples were separated and tested using Direct Agglutination Test (DAT) for detection of anti-L. infantum antibodies. Statistical analyses were performed using SPSS16. Results: Totally, 13 persons were seropositive; 7 samples with titer 1:800, 3 samples had 1:1600, 2 samples had 1:3200 and 1 sample had 1:6400. None of the seropositive cases had a history of kala-azar. There was no significant difference between males and females at titers of anti-Leishmania specific antibodies. Conclusion: L. infantum infection is being circulated with low prevalence in children up to 12 years old from SarPol-e-Zahab County but it is necessary that the surveillance system is regularly monitored among physicians and public health managers in the studied areas.

Live attenuated Leishmania infantum centrin deleted mutant (LiCen-/-) as a novel vaccine candidate: A field study on safety, immunogenicity, and efficacy against canine leishmaniasis.

This study was designed to evaluate the safety, immunogenicity, and efficacy of a single dose of L. infantum (LiCen-/-) live attenuated candidate vaccine against canine leishmaniasis (CanL). Eighteen healthy domestic dogs with no anti-Leishmania antibodies and negative leishmanin skin test (LST) were randomly inoculated intravenously with either L. infantum (LiCen-/-) vaccine candidate in 10 dogs or phosphate-buffered saline (PBS) in 8 dogs. The safety, immunogenicity, and efficacy rate of L. infantum (LiCen-/-) vaccine candidate against CanL were evaluated by different criteria, including clinical manifestations, injection-site lesion, hematology and biochemistry values, anti-Leishmania antibodies using direct agglutination test (DAT), delayed-type hypersensitivity (DTH) using LST, and CD4+ and CD8+ T-cells subsets, as well as by measuring interferon (IFN-γ), interleukin (IL-23), IL-17, and IL-10 cytokines. Spleen aspiration and detection of Leishmania parasite using parasitological examinations (microscopy and culture) were performed in both vaccinated and control groups. Two months after intervention, each dog was challenged intraperitoneally (IP) with wide type (WT) L. infantum. Two-month follow-up post vaccination showed no clinical signs and serious side effects associated with the vaccination. A significant increase was found in the expression of IL-17, CD4+, and CD8+ gene transcripts in PBMCs, as well as increased levels of Th1 cytokines, and reduction of Th2 cytokine. The efficacy of the vaccine candidate was calculated to be 42.85%. While the time window for assessing the vaccine's effectiveness was too limited to draw any real conclusions but the preliminary results showed a moderate efficacy rate due to inoculation a single dose of L. infantum (LiCen-/-) vaccine candidate. Further investigations with more sample sizes and multiple doses of the vaccine candidate using natural challenges in the endemic areas of CanL are recommended.

Diagnosis of Human Cutaneous Leishmaniasis: A Comparative Study Using CL Detect™ Dipstick, Direct Smear and Polymerase Chain Reaction Methods.

INTRODUCTION: In most of the endemic areas, the detection of CL is based on searching for amastigotes using the direct smear method. Since expert microscopists are not usually available in every laboratory, false diagnoses are a disaster that happens. Therefore, the aim of current research is to evaluate the validity of the CL Detect™ Rapid Test (CDRT) for diagnosis CL in comparison to direct smear and polymerase chain reaction (PCR) methods. METHODS: A total of 70 patients with skin lesions suspected to be CL were recruited. Skin samples from the lesions were collected and used for direct microscopic examination and the PCR method. Furthermore, the skin sample was collected in accordance with the manufacturer's instructions for the CDRT-based rapid diagnostic test. RESULTS: Of 70 samples, 51 and 35 samples were positive by direct smear examination and the CDRT, respectively. The PCR showed positive results in 59 samples; 50 and 9 samples were identified as Leishmania major and Leishmania tropica, respectively. The sensitivity and specificity were calculated to be 68.6% (95% CI 54.11-80.89%) and 100% (95% CI 82.35-100%). When the results of CDRT were compared to the microscopic examinations, an agreement of 77.14% was seen between the CDRT and microscopic examination. In addition, the sensitivity and specificity were 59.32% (95% CI 45.75-71.93%) and 100% (95% CI 71.5-100%) when the CDRT was compared to PCR assay (as gold standard) and an agreement (65.71%) was found between CDRT and PCR assay. CONCLUSION: As the CDRT is simple, rapid, and does not require great proficiency, it is recommended for use in the detection of CL caused by L. major or L. tropica as a diagnostic method, especially in areas with limited access to expert microscopists.

In Vitro Study on Four Types of Commercial Lectins on Leishmania infantum, L. major and L. tropica with Stage-Specific Binding and Leishmania Species Identification.

Background: We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. Methods: The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Results: Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 µg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Conclusion: Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

Seroepidemiological Study of Visceral Leishmaniasis (Kala-Azar) in Children under 12 Years Old in North of Iran: An Observational Study in 2019-2020.

Background: Visceral leishmaniasis (VL) caused by the Leishmania donovani complex that is transmitted by the bites of female sandflies. Mediterranean type of VL caused by L. infantum. While, Roudbar County of Guilan Province has been introduced as a suspected cutaneous leishmaniasis focus; there are no published data on the seroprevalence of VL in Guilan Province. We aimed to investigate the sero-prevalence of this disease in Roudbar County. Methods: This descriptive cross-sectional study was carried out in 2019-2020 among children less than 12 years of age to determine the seroprevalence of VL by direct agglutination test (DAT). Blood samples were randomly collected from 918 children under 12 years of age refers to the public health center in the clusters. Results: Out of 918 children, 14 (1.52%) showed anti-Leishmania antibodies, with 4 (0.43%), 2 (0.21%), 8 (0.87%) anti-L. infantum antibodies at titers 1:800, 1:1600 and ≥1: 3200 respectively. All children with anti-Leishmania antibody titers of ≥1:800 were evaluated by a physician. Clinical manifestation of VL including fever, anemia and hepatosplenomegaly observed in a 6-year-old boy from Defraz village with anti-Leishmania antibody of titers ≥102400.This patient was admitted to the pediatric hospital in Rasht, capital of Guilan province, Iran and was successfully treated. Conclusion: VL is being circulated with low prevalence in children up to 12 years old in Roudbar, northern part of Iran. Accordingly, it is critical to improve the awareness of physicians and public health supervisors about the importance of this fatal disease in Guilan province and especially in Roudbar area.

Visceral Leishmaniasis in Ardabil Province, Northwest of Iran: A Retrospective Epidemiological, Clinical and Paraclinical Study (1985-2018).

Background: We aimed to investigate the epidemiological, clinical and paraclinical aspects of Visceral Leishmaniasis in Ardabil Province, from 1986 to 2018. Methods: The disease has been diagnosed by a serological validate test as Direct Agglutination Test, parasitological methods, and clinical manifestations. The data were collected and then analyzed. Results: Overall, 2824 out of 69007cases (4.1%) had anti-Leishmania antibodies at titers≥1:3200. The majority of VL cases belonged to Meshkin-Shahr, and then Germi County. The percentages of both male and female cases were 52.8 % and 46.6%, respectively. Approximately 78.1% of cases were less than 5 yr old. The high incidence rate of the disease was recorded in the years 1987 and 2003. 48.6%, 3.2%, and 1.9% of the patients had low titer (1:3200), high titer (1:102400), and suspicious titer (1:1600), respectively. Out of 1200 bone marrow aspiration, 23% were positive, and 77% were negative. Fever, paleness, and hepatosplenomegaly were the main symptoms in patients. Moreover, abdominal swelling, edema, weight loss, lymphadenopathy and jaundice were some other symptoms in the patients. Among biochemistry factors, the levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, triglyceride and cholesterol have been increased. The death rate was estimated to be 2.8%, mainly related to the Ardabil and Germi cases. Conclusion: Kala-Azar is still one of the most important infectious diseases in Ardabil Province, and is responsible for considerable death rate in infants. More attention should be done to parasite control and early diagnosis.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

BACKGROUND: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. OBJECTIVES: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. METHODS: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. RESULTS: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. CONCLUSIONS: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts.

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

A Convenient and Sensitive kDNA-PCR for Screening of Leishmania infantum Latent Infection Among Blood Donors in a Highly Endemic Focus, Northwestern Iran.

BACKGROUND: Recent global evidences showed that asymptomatic blood donor carriers of Leishmania infection will appear as a threat for blood transfusions recipients in endemic areas. As yet, there is no appropriate diagnostic procedure for detecting infection of blood donors in blood banks. SUBJECTS AND METHODS: The present study was aimed to apply various current diagnostic tests among blood donors in an endemic area of visceral leishmaniasis (VL), Ardabil Province, northwestern Iran. Blood samples were gathered from 860 blood donors in endemic areas of the province between 2017 and 2018, at eight blood donation centers. These samples was assessed using microculture, serological (DAT and rK39-ICT) and molecular based (conventional kDNA-PCR and HRM-PCR) tests. RESULTS: Of 860 eligible donors, 24 (2.8%) were seropositive for VL by DAT, and 388 (45%) were positive by kDNA-PCR. Moreover, 19 (19/860) were positive for both of them. Out of 19 subjects, 5.3% (1/19) was positive by rK39-ICT, 10.5% (2/19), and 79% (15/19) were detected positive in microculture and HRM-PCR methods, respectively. Nineteen donors were followed up for 2 years, of which 16 (84.2%) had a serological conversion, and 4 (21%) were positive by kDNA-PCR. The sensitivity of kDNA-PCR, and HRM-PCR procedures in detecting Leishmania parasite was found to be 98.7%, and 79%, respectively. CONCLUSIONS: Our findings justify the use of kDNA-PCR as a convenient and sensitive tool for screening subjects with leishmanial latent infection in blood banks at least in endemic regions. In these areas, however, a PCR-based test should be used to validate Leishmania infection among seropositive donors.

A Sero-Epidemiological Study on Visceral Leishmaniasis among Volunteer Children and Adults in Rural Areas of Shahroud, Iran 2018-2019.

Background: Visceral leishmaniasis (VL) also known as Kala-azar is considered as one of the zoonotic infections in Mediterranean countries. The disease reservoir and vectors are dogs and sandflies respectively. Due to reported sporadic cases of Kala-azar in the past five years in Shahroud County, Semnan Province, Iran, this study aimed to investigate the status of this infection in this area and to determine its seroepidemiology to take required measurements for infection control and treatment. Methods: This study was conducted on 504 subjects residing in seven villages in Shahroud County. Blood samples were randomly selected using the cluster sampling method and were collected from subjects aged up to 13 years old (90%) and adults over 13 years old (10%) from September to May 2019. After separating sera from whole blood, samples were subjected to direct agglutination test (DAT) to detect anti-Leishmania infantum antibodies. A range of 1:10 to 1:800 dilutions were prepared from the samples. Results: Results of 1:800 titration indicated that no sample was positive for antibodies against L. infantum. After the secondary screening, 10 cases (1.98%) showed the antibody titer of 1:100, while four cases (0.79%) showed the antibody titer of 1:400. Of 14 cases with the L. infantum antibodies, all were detected from the children <13 years old. According to clinical findings, no patient was suffering from fever, weight loss, splenomegaly, hepatomegaly, and cachexia and therefore did not show the Kala-azar symptoms. Conclusion: The results of the current study indicate that Kala-azar is not prevalent in Shahroud County.

Designing and Evaluation of a Recombinant Multiepitope Protein by Using ELISA for Diagnosis of Leishmania infantum Infected in Dogs.

BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Correct identification of infected patients and reservoirs is vital to control the spread of VL. One important step in the control of Zoonotic Visceral leishmaniasis (ZVL) is the identification of infected dogs, which are the main domestic reservoir hosts of Leishmania infantum. We aimed to prepare and evaluate a new recombinant antigen using Bioinformatics tools for diagnosis of ZVL in domestic dogs. METHODS: The present study was carried out in Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran during 2015-2018. Three L. infantum (JPCM5 strain) proteins were analyzed as follows: Nucleotide sequences of the surface proteins, putative amastin-like surface protein (P1), surface antigen protein 2 precursor (P2) and surface antigen-like protein (P3). The epitopes were predicted by several different bioinformatics servers using different methods. The predicted epitopes were selected with the highest immunogenic potential (P1P2P3) linked to each other with linkers (Gly, Se) and synthesized. Then the expression and protein purification were performed. In total, 114 serum samples were collected at 7 months. Positive and negative sera were confirmed using direct agglutination test (DAT). These recombinant antigens from L. infantum were used by indirect ELISA. RESULTS: Considering the cut-off point of 0.23, the test showed a sensitivity of 98% (95%CI=89.50%-99.90%) and a specificity of 95.31% (95%CI=87.10%-98.72%). Kappa analysis indicated very good agreement (kappa=0.831) between ELISA and DAT (P<0.05). CONCLUSION: ELISA using the recombinant protein P1P2P3 has great potential for the diagnosis of canine visceral leishmaniasis (CVL).

A comparative study of nested-PCR and direct agglutination test (DAT) for the detection of Leishmania infantum infection in symptomatic and asymptomatic domestic dogs.

OBJECTIVE: Canine visceral leishmaniasis (CVL) is the main source of human visceral leishmaniosis (HVL) in Mediterranean region, including Iran and is spread from domestic dogs to Phlebotomine sand flies vectors to humans. To control the transmission of HVL, early and accurate detection of infected dogs is paramount importance despite it remains a confronting challenge. Herein, we evaluated the performance of direct agglutination test (DAT) against gold standard nested polymerase chain reaction (nested-PCR) for CVL diagnosis in symptomatic and asymptomatic domestic dogs from endemic areas of Iran. RESULTS: Venous blood samples were collected from dogs without clinical signs (n = 30) and with clinical signs (n = 35) suggestive of Leishmania infantum infection. Among 65 samples examined, Leishmania DNA was detected by nested-PCR in 89.23% (58/65). Furthermore, 86.15% (56/65) nested-PCR positive samples were also DAT positive. The results of the DAT sensitivity test were 96.43% and 96.67% in symptomatic and asymptomatic dogs, respectively, while the specificity was 100.00% and 60.00% in symptomatic and asymptomatic dogs, respectively. The results of this study also pointed out substantial concordance between DAT test and nested-PCR method in both symptomatic dogs (Κ = 0.783; P < 0.001) and asymptomatic dogs (Κ = 0.618; P < 0.001). Thus, DAT represents as a simple and economic tool for initial diagnosis of CVL particularly in endemic areas of the disease.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

The diagnostic accuracy of direct agglutination test for serodiagnosis of human visceral leishmaniasis: a systematic review with meta-analysis.

BACKGROUND: Direct agglutination test (DAT) as a simple, accurate and reliable method, has been widely used for serodiagnosis of visceral leishmaniasis (VL) during the last three decades. The present study is a systematic review and meta-analysis to evaluate the diagnostic accuracy of DAT for serodiagnosis of human VL. METHODS: Electronic databases, including MEDLINE (via PubMed), SCOPUS, Web of Science, SID and Mag Iran (two Persian scientific search engines) were searched from December 2004 to April 2019. We determined the pooled sensitivity and specificity rates of DAT for the diagnosis of human VL, calculated positive and negative likelihood ratios (LR+ and LR-), and constructed summary receiver operating characteristic (ROC) curves parameters across the eligible studies. RESULTS: Of the 2928 records identified in the mentioned electronic databases and after examining reference lists of articles, 24 articles met inclusion criteria and were enrolled in the systematic review and out of them 20 records qualified for meta-analysis. The pooled sensitivity and specificity rates of DAT was 96% [95% CI, 92-98] and 95% [CI95% 86-99], respectively. The likelihood ratio of a positive test (LR+) was found to be 21 [CI95%, 6.6-66.5] and the likelihood ratio of a negative test (LR-) was found to be 0.04 [(CI95%, 0.02-0.08]. The combined estimate of the diagnostic odds ratio for DAT was high [467 (CI95%, 114-1912]). We found that the summary receiver operating characteristic curve (SROC) is positioned near the upper left corner of the curve and the area under curve (AUC) was 0.98 (95% CI, 0.97 to 0.99). CONCLUSION: Referring to our analysis, we determined that DAT can be considered as a valuable tool for the serodiagnosis of human VL with high sensitivity and specificity. As DAT is a simple, accurate and efficient serological test, it can be recommended for serodiagnosis of human VL particularly in endemic areas.

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis in Owned Dogs (Canis familiaris) in New Foci of Rural Areas of Alborz Province, Central Part of Iran: A Cross-Sectional Study in 2017.

BACKGROUND: Mediterranean form of visceral leishmaniasis (VL) is endemic among some provinces of Iran. The present study was designed to determine the prevalence of canine visceral leishmaniasis (CVL) in the owned dogs of the rural areas of Alborz Province near Tehran as the capital of Iran. METHODS: This study conducted on 303 owned dogs that selected using a stratified random sampling method. The direct agglutination test (DAT) was used to determine the frequency of Vl. The spleen biopsy was taken from the serology-positive dogs for the confirmation of CVL in the suspected dogs. Nested PCR and sequencing methods were used to determine the type of Leishmania species in the dogs which were parasitological positive. RESULTS: Overall, the DAT results of 9 dogs (2.97%, CI: 1.57-5.55) showed anti Leishmania antibodies at titers ≥ 1:320 indicating VL infection. One dog (0.33%, CI 95%: 0.06-1.85) showed clinical signs and symptoms of VL. There was a significant correlation between the positive cases of CVL and rural area (p< 0.001). The Leishmania was observed in the impression smears that were prepared from spleen biopsy of five the studied dogs. Leishmania infantum were confirmed in all them using nested-PCR assay. The sequence analysis of all five isolates was 95% similar to L. infantum. CONCLUSION: This study shows that domestic cycle of L. infantum has been established in rural areas of Alborz province where located near Tehran as capital city of Iran. It is necessary to increase the awareness and monitoring of the disease periodically.