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.

High occurrence of Toxoplasma gondii infection among blood donors in Ardabil Province as main focus of zoonotic visceral leishmaniosis, northwestern Iran.

Toxoplasmosis, as cosmopolitan parasitic disease, is considered as one of the transfusion-transmitted parasites. The true burden of Toxoplasma gondii (T. gondii) amongst blood donors remains undisclosed around the world. Since there was no evidence on the prevalence of T. gondii in blood donors in Ardabil Province, as main focus of zoonotic visceral leishmaniosis (ZVL), northwestern Iran, current research was therefore conducted to estimate the seroprevalence of T. gondii and PCR assay among them for the first time. In the present study, 462 plasma samples from asymptomatic blood donors of Ardabil Province, northwestern Iran, were tested for IgM and IgG anti-T. gondii antibodies levels using ELISA test. Moreover, the buffy coat of all seropositive subjects was screened for T. gondii DNA by conventional PCR. Also, the data sheet consisting of characteristic information was registered for all the applicants. Overall, anti-T. gondii antibodies were found in 36% (166/462) of asymptomatic blood donors. Anti-T. gondii IgM and IgG seroprevalence was 1.5% and 32.5%, respectively. Only nine subjects (2%) were found to be positive for both IgM and IgG. Moreover, T. gondii DNA was identified in 18% (30/166) of seropositive donors. The logistic regression analysis showed a significant correlation between T. gondii seropositivity and contact with cats, agricultural activities, history of consumption of undercooked meat and being non-educated (P=0.001). The high prevalence (about one-third) of anti-T. gondii antibodies and possibly active infection using conventional PCR test represents that asymptomatic carriers of T. gondii are quite common in the study areas and pose a potential threat to the blood safety and hemovigilance program.

Actin Gene-Based Molecular Typing of Trichomonas vaginalis Clinical Isolates from the North of Iran.

AIM: The aim of the current study is to evaluate the prevalence of trichomoniasis in men and women in the north of Iran and to find genotypes in the positive clinical specimens based on T. vaginalis actin gene. MATERIALS AND METHODS: Women's genital (n = 500) and men's urine (n = 1500) samples were collected from the participants referred to clinics in Mazandaran Province, northern Iran, during 2006-2018. In addition, 1500 Pap smear specimens, archived in the Bu Ali Hospital, Sari City, Mazandaran Province, northern Iran, were examined. The specimens were examined based on parasitological methods, nested polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism, and phylogenetic analysis. RESULTS: Overall, 17 (0.48%) of 3500 specimens were positive by PCR. Total prevalence was 0.55% (n = 2000) for women, of which 500 (1.4%; n = 7) specimens were collected freshly, and 1500 (0.26%; n = 4) were Pap smears. Moreover, six (0.4%) out of 1500 men urine specimens were positive. Overall, genotypes G, E, and I were detected with the prevalence of seven (0.2%), seven (0.2%), and three (0.08%), respectively. There was no significant statistical difference among the prevalence of the detected genotypes (P > 0.05). CONCLUSION: As a whole, the prevalence of trichomoniasis was low in the studied area in the north of Iran and, most importantly, the genotypes of E, G, and I were distributed among men and women in the province.

Hepcidin gene polymorphisms and iron overload in ß-thalassemia major patients refractory to iron chelating therapy.

BACKGROUND: ß Thalassemia is one of the most common groups of hereditary haemoglobinopathies. Affected people with thalassemia major are dependent on regular blood transfusion which on the long term leads to iron overload. Hepcidin is a peptide hormone and an important regulator of iron homeostasis, especially in thalassemia. Expression of this hormone is influenced by polymorphisms within the hepcidin gene, HAMP. Several studies emphasized the role of single nucleotide polymorphisms (SNPs) located in the promoter region of the gene. This study aimed to analyze the association between three SNPs in promoter of HAMP, c.-582A > G, c.-443C > T, and c.-153C > T, with iron overload in ß-thalassemia major patients. METHODS: A total of 102 samples from ß thalassemia major patients were collected. Genomic DNA was extracted and segments of DNA encompassing rs10421768 and rs142126068 were sequenced. Statistical analysis was performed by SPSS Statistics 23 using independent t test and Fisher's exact test. RESULTS: A total of 102 adult ß-thalassemia major patients were genotyped for three SNPs in the promoter region of HAMP gene by PCR and direct sequencing. Most of the patients (71.3%) were iron overloaded (based on plasma ferritin > 1000 ng/ml) in spite of receiving regular iron-chelating therapy. Our analysis revealed a statistically significant difference between the level of cardiac iron accumulation and c.-582A > G variant (p = 0.02). For c.-443C > T statistical analysis was on the edge of the significant relationship between the minor allele and serum ferritin (p = 0.058). All samples were homozygous for allele C of c.-153C > T. CONCLUSIONS: Despite chelating therapy, iron overload is still one of the main complications of thalassemia. Our findings and others emphasize the role of hepcidin -582A > G polymorphism as a key component of iron homeostasis in these patients.

Transfusion reactions: A retrospective analysis of the Iranian national haemovigilance system (INHS) data.

BACKGROUND: The use of blood and blood components is an essential and effective treatment for many patients, but the transmission of infectious agents via transfusion and the occurrence of non-infectious transfusion reactions (TRs) are unavoidable. The objective of our study was to determine the frequency and type of transfusion-related reactions reported in Iran between 2014 and 2018. METHODS: This retrospective study was carried out in the Iranian blood transfusion organization (IBTO) during a period of five years (2014-2018). All TRs reported to the Iranian national haemovigilance system (INHS) were analyzed using SPSS software. RESULTS: A total of 20,062 TRs were reported to the INHS from 2014 to 2018. The overall frequency of TRs was 0.14 %. The most common TRs were allergic (42.51 %) and febrile non-hemolytic reactions (37.17 %), respectively. The frequency of TRs to red blood cell (RBC) components was significantly higher than those to the components of platelet (PLT) and plasma (P < 0.05). CONCLUSIONS: In the present study, the frequency of TRs was relatively low (0.14 %) and the majority of them were allergic and febrile non-hemolytic reactions. The recording and reporting of all occurred TRs, the implementation of INHS in all hospitals and the continuity of specific educational courses to physicians, nurses and blood banking staff, as well as the use of an online reporting system will help to improve the haemovigilance in Iran.

Prevention strategies of transfusion-transmitted parasitic infections (TTPIs): Strengths and challenges of current approaches, and evaluation of the strategies implemented in Iran.

BACKGROUND: Several strategies are being implemented in blood transfusion centers of the world to prevent the transfusion-transmitted parasitic infections (TTPIs). The objective of this study was to determine and describe the strategies to minimize the transmission risk of parasitic agents via blood transfusion in Iran. METHODS: This study was conducted in the Iranian blood transfusion organization (IBTO). The data were extracted from the latest version of the "medical interview" standard operating procedure (SOP). RESULTS: The donor selection is the first and only step to reduce the risk of TTPIs in endemic and non-endemic areas of Iran. In all blood transfusion centers of the IBTO, the blood donation volunteers with a previous history of malaria, Chagas disease, visceral leishmaniasis (VL), muco-cutaneous leishmaniasis and babesiosis, as well as those with clinical toxoplasmosis, cutaneous leishmaniasis (CL) and with a history of residence in, or travel to, malaria-endemic areas are permanently or temporarily deferred from the blood donation. CONCLUSIONS: Since malaria, toxoplasmosis and VL are endemic in parts of Iran, as well as the increasing travels to endemic areas and immigrations from endemic to non-endemic areas of parasitic infections, the extensive use of blood and blood components and the asymptomatic occurrence of most parasitic infections in blood donors, the donor selection strategy is not sufficient to prevent the TTPIs. Therefore, the changing of donor selection process and the use of other common preventive strategies are recommended to reduce the risk of TTPIs, especially for high-risk groups of toxoplasmosis and VL.

Frequency and genetic diversity of Blastocystis subtypes among patients attending to health centers in Mazandaran, northern Iran.

Blastocystis hominis is a common intestinal protozoan with global distribution. The prevalence of B. hominis is extremely diverse in different countries. Molecular studies show that there is a great deal of genetic variation in the Blastocystis parasite isolated from humans and animals. Therefore, this study was conducted to determine B. hominis genotypes in patients attending to health centers of Sari, Mazandaran Province, north of Iran. 420 fecal specimens were collected from individuals in health centers of Sari from April to December 2017. The samples were examined by direct methods, parasite test (formalin ether) and Giemsa staining. In order to determine the genotypes of Blastocystis parasite, all positive samples in direct methods were tested by PCR using SSU rDNA gene. Eventually, seven positive molecular samples were sequenced and recorded in the gene bank. The phylogenetic analysis was done by drawing a gene tree using the neighbor-joining procedure. B. hominis was detected in 60 stool specimens by microscopic method. All these 60 samples were positive by PCR and the bands 310 bp were observed. According to the phylogenetic tree, it was revealed that B. hominis isolates of Mazandaran Province are more similar to subtype ST3 (ST type) that reported in Khorramabad in Lorestan Province (West of Iran). The present study showed that, like most studies, ST3 is the dominant genotype of protozoan in Sari. Improvement of public health awareness, relative improvement in environmental health, urban wastewater treatment, proper clothing coverage and the use of gloves during dealing with livestock and the use of domestic water treatment devices can reduce the Blastocystis infection in this area.

Blood Donor Deferrals for Malaria in Iran: A Five-Year Retrospective Study.

BACKGROUND: Malaria is one of the most important transfusion-transmitted infections (TTIs) worldwide. To prevent the occurrence of transfusion-transmitted malaria (TTM), potential blood donors with a history of malaria or travel to, or residence in, malarious areas are permanently or temporarily deferred from donating blood. The objective of the present study was to evaluate the blood donor deferrals for malaria in Iran. METHODS: This descriptive cross-sectional study was conducted in the Iranian Blood Transfusion Organization (IBTO) from 21 Mar 2011 to 19 Mar 2016 (5 yr). The data were collected and extracted from IBTO comprehensive database, namely Negareh, and then recorded in a structured template form. Statistical analysis was performed using SPSS. RESULTS: Of the 12,790,859 blood donation volunteers, 23,084 (0.18%) were deferred due to the risk of malaria. More than 90% of malaria-deferrals were because of travel to and residence in malaria endemic areas. Among the malaria-deferred volunteers, 22,139 (95.91%) were male and 945 (4.09%) were female; 2,053 (8.89%) were permanently deferred, while 21,031 (91.11%) were temporarily deferred. The highest malaria-deferral rates were observed in South Khorasan (0.82%), Razavi Khorasan (0.79%) and Yazd (0.54%) provinces, respectively. CONCLUSION: Given the prevalence of malaria in neighboring countries (Pakistan and Afghanistan) and several provinces of Iran and the increasing human migration and movement between malaria non-endemic and endemic areas, the malaria-deferral rate might be higher than 0.18% in Iran. Thus, the changing, as well as the precise and accurate implementation of donor selection process must be considered in all blood transfusion centers of Iran.

Genetic diversity at the C-terminal domain of knob-associated histidine-rich protein (KAHRP) of Plasmodium falciparum isolates from Burundi, Eastern Africa.

The knob-associated histidine-rich protein (KAHRP) is an exported parasite protein and the major component of infected erythrocytes by Plasmodium falciparum. P. falciparum histidine-rich protein-1 (PfHRP-1) is docked by KAHRP, which this interaction plays a significant role in cytoadherence of the malaria protozoan to erythrocytes and pathogenicity. The most polymorphic region of the PfHRP-1 is the C-terminal of decapeptide repeat domain (region III). The main objective of this study was to explore the genetic diversity at the region III of KAHRP in P. falciparum isolates from Burundi. In the present study, the nested PCR was performed for the amplification of the coding gene (kahrp gene) for region III in 35 P. falciparum isolates from Burundi. The nested PCR products of seven randomly selected isolates were purified and then sequenced. As the result, three allelic forms (340 bp, 370 bp, and 400 bp) were seen at the C-terminal domain of kahrp gene. The existence of multiple alleles of the kahrp gene revealed the presence of different P. falciparum strains in Burundi. It is suggested that the results could be useful in designing and the improvement of targeted therapy agents for falciparum malaria.

Toxoplasma gondii in Blood Donors: A Study in Boyer-Ahmad County, Southwest Iran.

Toxoplasma gondii is an important foodborne protozoan that can be transmitted through infected blood containing tachyzoite form of the parasite. The current study aimed to evaluate the prevalence of T. gondii infection and related risk factors among healthy blood donors in Boyer-Ahmad County, southwest Iran. Blood samples were taken from 285 healthy blood donors who voluntarily agreed to participate in this study. Sera and buffy coat were isolated from the blood samples for serological and molecular evaluations. The sera were tested for anti-T. gondii antibodies (both IgG and IgM), using a commercial ELISA kit. The buffy coat of seropositive cases was evaluated for detection of T. gondii DNA by PCR. Moreover, a structured questionnaire, containing socioepidemiological data and possible risk factors, was filled out by each participant during sample collection. Anti-T. gondii antibodies were detected in sera of 48/285 (16.8%) participants. Only two of the subjects (0.7%) were seropositive for both IgG and IgM antibodies. T. gondii DNA was not detected in buffy coat of any of the seropositive cases. Risk factors such as contact with soil (OR, 9.7; 95% CI, 4.9-19.4) and consumption of semicooked meat (OR, 2.5; 95% CI, 1.2-5.03) were statistically associated with seropositivity to T. gondii. The seroprevalence rate of T. gondii antibodies in the blood donors of Boyer-Ahmad County was not high in comparison with other regions in Iran. In this study, consumption of undercooked meats, job, and contact with soil were independent risk factors associated with T. gondii infection, which can be considered as potential sources of T. gondii infection.

Global status of visceral leishmanial infection among blood donors: A systematic review and meta-analysis.

INTRODUCTION: Transmission of Leishmania through transfusion has been reported from various Visceral leishmaniasis (VL) endemic areas of the world. The true burden of Leishmania infection in blood donors remains generally unknown. Thus, the present systematic review attempted to determine the global prevalence of Leishmania infection among blood donors. METHODS: Data were extracted through five English and five Persian databases during the period from 1997 to 2016. Overall, 16 articles fulfilled the inclusion criteria and were used for data extraction in this systematic review. RESULTS: In total, 13,743 blood donors from different regions of world were examined. The prevalence rate of Leishmania infection according to seropositivity obtained 7% (95%CI: 5%, 8%). The lowest and the highest prevalence were related to Bangladesh 0.25% (95%CI: 0.0%, 1.0%) and Brazil, 16% (95%CI: 12%, 19%). Seroprevalence rate of leishmaniasis among females was more (4.60%) than males. Of 15 studies included in the meta-analysis, the pooled prevalence rate of molecular tests was obtained 2% (95%CI: 1%, 3%) in which Iran and Spain had the lowest and the highest prevalence, 0.05% and 7%, respectively. Our analysis showed that L. infantum was more common than L. donovani as etiological agent of VL among all donors. CONCLUSION: Our data confirms the presence of asymptomatic carriers of VL in endemic areas and supplies as an attentive to the likelihood of these carriers acting as blood donors. Moreover, we conclude that molecular tests for screening in asymptomatic blood donor provide an accurate estimate of the rate of infection over serological tests.

Asymptomatic human blood donors carriers of Leishmania infantum: Potential reservoirs for visceral leishmaniasis in northwestern Iran.

BACKGROUND: Little is known regarding transfusion-transmitted leishmaniasis (TTL) and the real global incidence of visceral leishmaniasis (VL) among blood donors as carriers of Leishmania spp. in endemic VL foci including Iran. Recent epidemiological evidences indicate that blood donor may be harbor of Leishmania infantum (L. infantum) infection in Iran. METHODS: The aim of the present study was to determine the prevalence of Leishmania infection among blood donors in a main endemic focus of VL in Iran using DAT and polymerase chain reaction (PCR) based methods Between July-September 2016, blood samples were collected from 600 apparently healthy blood donors from six blood donation centers and blood donation mobile unit in Ardabil Province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination test (DAT), and for L. infantum kDNA, the PCR-based assay. RESULTS: Of 600 blood donors, which were examined, 23 (3.8%) blood donors were seropositive by DAT and of 23 seropositive subjects, 82.6% (19/23) were positive by PCR. All the seropositive cases were males except one of them was female. CONCLUSIONS: Our findings showed that many asymptomatic human carriers of L. infantum live in the endemic regions of northwestern Iran and potentially act as reservoirs of infection; those must be considered carefully by arrangement VL control strategies in the country.

Prevalence of Toxoplasma gondii among Iranian Blood Donors: A Narrative Review Article.

BACKGROUND: Toxoplasmosis is a common parasitic disease. There is likelihood of exposure to Toxoplasma gondii in blood donors during the periods of life. Currently, laboratory screening of blood donors for T. gondii is not routinely available. The objectives of this review were to study the effects of T. gondii on blood safety and to approach for risk reduction in blood recipients. METHODS: A literature search was performed using Cochrane library, PubMed, Scopus, Google scholar IranMedex, SID and Magiran without time limitation. All studies, which had reported the prevalence of T. gondii in Iranian blood donors in both English and Farsi languages, were evaluated and reviewed. The contents of the transfusion medicine text books related to this issue were reviewed. Searching keywords were "Blood Donors" or "Blood Transfusion" and "Toxoplasma" or "Toxoplasmosis" and Iran. RESULTS: In order to study the prevalence of T. gondii in Iranian blood donors, six studies have been reviewed. IgG and IgM antibodies varied between 12.3% to 52.8% and 0% to 5.47%. Some of these studies have suggested to doing the screening for all blood donors. However, based on parasitological and epidemiological evidences, there is little chance for parasite transmission by blood transfusion. CONCLUSION: By considering the moderate prevalence, difficulty in the differentiation between recent and past infections, and cost-effectiveness, it is not possible and rational to perform screening of donated blood. To reduce the risk of parasite transmission, leukofilteration method is recommended.

Transfusion-Transmitted Malaria in Iran: A Narrative Review Article.

BACKGROUND: Malaria is the most important transfusion-transmitted infection (TTI) in worldwide after viral hepatitis and human immunodeficiency virus (HIV) infection. The main objective of the present study was to review and evaluate the transmission of malaria via blood transfusion in Iran. METHODS: A literature search was done without time limitation in the electronic databases as follows: PubMed, Scopus, Google Scholar, Web of Science, Science Direct, scientific information database (SID), Magiran, IranMedex and Irandoc. The searches were limited to the published papers to English and Persian languages. RESULTS: Six papers were eligible. From 1963 to 1983, 344 cases of Transfusion-transmitted malaria (TTM) had been reported from different provinces of Iran. The most prevalent species of involved Plasmodium in investigated cases of TTM was Plasmodium malariae (79.24%). The screening results of 1,135 blood donors for malaria were negative by microscopic examination of peripheral blood smears and rapid diagnostic test (RDT) methods. CONCLUSION: Lack of TTM report from Iran in the last three decades indicates that the screening of blood donors through interviewing (donor selection) may be effective in the prevention of the occurrence of transfusion-transmitted malaria.

Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran.

The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.

Genetic polymorphism at the C-terminal domain (region III) of knob-associated histidine-rich protein (KAHRP) of Plasmodium falciparum in isolates from Iran.

The knob-associated histidine-rich protein (KAHRP) plays a major role in the virulence of Plasmodium falciparum and is one of the targets for molecular therapy. The primary structure of KAHRP of P. falciparum consists of three domains (regions I-III), of which the C-terminal domain (region III) is the most polymorphic segment of this protein. One of the main obstacles is genetic diversity in designing and developing of malaria control strategies such as molecular therapy and vaccines. The primary objective of the present study was to investigate and analyze the extent of genetic polymorphism at the region III of KAHRP of P. falciparum in isolates from Iran. A fragment of the kahrp gene spanning the C-terminal domain was amplified by nested PCR from 50 P. falciparum isolates collected from two malaria endemic areas of Iran during 2009 to August 2010 and sequenced. In this study, three allelic types were observed at the C-terminal domain of KAHRP on the basis of the molecular weight of nested PCR products and the obtained sequencing data. The presence of multiple alleles of the kahrp gene indicates that several P. falciparum strains exist in the malaria endemic areas of Iran. Our findings will be valuable in the design and the development of the molecular therapeutic reagents for falciparum malaria.