Electrochemical quantitation of Leishmania infantum based on detection of its kDNA genome and transduction of non-spherical gold nanoparticles.

Detecting and monitoring the pathogens with high selectivity and sensitivity is critical for public health. In the present study, we demonstrated a specific analytical strategy for sensitive detection of Leishmania infantum genome. The developed sensor utilized toluidine blue as a hybridization indicator and a Leishmania infantum-specific capture DNA sequence immobilized on a high-surface area gold nanostructure as an electrochemical transducer. The produced analytical response was based on the hybridization of the single-stranded DNA from the target with the immobilized DNA sequence at the electrode surface. The developed DNA sensor in this study was successfully employed to detect a synthetic Leishmania infantum target sequence in a wide concentration range from 1 × 10-18 to 1 × 10-10 mol L-1 with a detection limit of 0.2 amol L-1 with the ability to discriminate the target sequence from mismatched sequences. Moreover, the designed DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles. The DNA sensor could detect Leishmania infantum genome in a wide concentration range from 15 to 50 ng µL-1 with a detection limit of 29 ng µL-1. Furthermore, clinical trials confirmed the applicability of the developed DNA sensor for practical applications.

Evaluation of the Effect of Radiofrequency Radiation Emitted From Wi-Fi Router and Mobile Phone Simulator on the Antibacterial Susceptibility of Pathogenic Bacteria Listeria monocytogenes and Escherichia coli.

Mobile phones and Wi-Fi radiofrequency radiation are among the main sources of the exposure of the general population to radiofrequency electromagnetic fields (RF-EMF). Previous studies have shown that exposure of microorganisms to RF-EMFs can be associated with a wide spectrum of changes ranged from the modified bacterial growth to the alterations of the pattern of antibiotic resistance. Our laboratory at the nonionizing department of the Ionizing and Non-ionizing Radiation Protection Research Center has performed experiments on the health effects of exposure to animal models and humans to different sources of electromagnetic fields such as cellular phones, mobile base stations, mobile phone jammers, laptop computers, radars, dentistry cavitrons, magnetic resonance imaging, and Helmholtz coils. On the other hand, we have previously studied different aspects of the challenging issue of the ionizing or nonionizing radiation-induced alterations in the susceptibility of microorganisms to antibiotics. In this study, we assessed if the exposure to 900 MHz GSM mobile phone radiation and 2.4 GHz radiofrequency radiation emitted from common Wi-Fi routers alters the susceptibility of microorganisms to different antibiotics. The pure cultures of Listeria monocytogenes and Escherichia coli were exposed to RF-EMFs generated either by a GSM 900 MHz mobile phone simulator and a common 2.4 GHz Wi-Fi router. It is also shown that exposure to RF-EMFs within a narrow level of irradiation (an exposure window) makes microorganisms resistant to antibiotics. This adaptive phenomenon and its potential threats to human health should be further investigated in future experiments. Altogether, the findings of this study showed that exposure to Wi-Fi and RF simulator radiation can significantly alter the inhibition zone diameters and growth rate for L monocytogenes and E coli. These findings may have implications for the management of serious infectious diseases.

A label-free, PCR-free and signal-on electrochemical DNA biosensor for Leishmania major based on gold nanoleaves.

Detection of leishmaniasis is important in clinical diagnoses. In the present study, identification of Leishmania parasites was performed by a label-free, PCR-free and signal-on ultrasensitive electrochemical DNA biosensor. Gold nanoleaves were firstly electrodeposited by an electrodeposition method using spermidine as a shape directing agent. The biosensor was fabricated by immobilization of a Leishmania major specific DNA probe onto gold nanoleaves, and methylene blue was employed as a marker. Hybridization of the complementary single stranded DNA sequence with the biosensor under the selected conditions was then investigated. The biosensor could detect a synthetic DNA target in a range of 1.0×10-10 to 1.0×10-19molL-1 with a limit of detection of 1.8×10-20molL-1, and genomic DNA in a range of 0.5-20ngµL-1 with a limit of detection of 0.07ngµL-1. The biosensor could distinguish Leishmania major from a non-complementary-sequence oligonucleotide and the tropica species with a high selectivity. The biosensor was applicable to detect Leishmania major in patient samples.

An electrochemical genosensor for Leishmania major detection based on dual effect of immobilization and electrocatalysis of cobalt-zinc ferrite quantum dots.

Identification of Leishmania parasites is important in diagnosis and clinical studies of leishmaniasis. Although epidemiological and clinical methods are available, they are not sufficient for identification of causative agents of leishmaniasis. In the present study, quantum dots of magnetic cobalt-zinc ferrite (Co0.5Zn0.5Fe2O4) were synthesized and characterized by physicochemical methods. The quantum dots were then employed as an electrode modifier to immobilize a 24-mer specific single stranded DNA probe, and fabrication of a label-free, PCR-free and signal-on electrochemical genosensor for the detection of Leishmania major. Hybridization of the complementary single stranded DNA sequence with the probe under the selected conditions was explored using methylene blue as a redox marker, utilizing the electrocatalytic effect of the quantum dots on the methylene blue electroreduction process. The genosensor could detect a synthetic single stranded DNA target in a range of 1.0×10(-11) to 1.0×10(-18)molL(-1) with a limit of detection of 2.0×10(-19)molL(-1), and genomic DNA in a range of 7.31×10(-14) to 7.31×10(-6)ngµL(-1) with a limit of detection of 1.80×10(-14)ngµL(-1) with a high selectivity and sensitivity.

Comparative study on isoenzyme patterns of Fasciola hepatica and Fasciola gigantica.

Differences in the number of isoenzymes and their electrophoretic mobility are very valuable tools for characterization of closely related species of a certain parasite. This study aimed to compare the isoenzymatic patterns of Fasciola hepatica and Fasciola gigantica. Adult F. hepatica and F. gigantica were collected from infected livers of sheep and cattle and their species was determined by molecular method. Enzymes were extracted from the adult worms and subjected to electrophoresis through a polyacrylamide gel. The activities of nine enzymatic systems; including superoxide dismutase (SOD), glucose phosphate isomerase (GPI), glucose 6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), malic enzyme (ME), nucleoside hydrolyse 1 (NH1), phosphoglucomutase (PGM), isocitrate dehydrogenase (ICD), and 6-phosphogluconate dehydrogenase (6PGD) were evaluated from both Fasciola species. The enzymatic profile obtained for SOD system for F. hepatica were two-banded pattern whereas in F. gigantica it was a four-banded pattern. NH1 revealed one band in F. hepatica with relative mobility of 0.05 and two bands in F. gigantica with relative mobility of 0.05 and 0.66. In ICD, GPI and G6PD enzyme systems, both Fasciola species revealed one band but with different relative mobility. Isoenzymatic profile of MDH, 6PGD, PGM and ME were the same in both species. Findings of this study revealed that F. hepatica and F. gigantica have entirely different isoenzyme patterns in the enzymes of ICD, G6PD, GPI, PGM and SOD. These enzyme systems may be used for differentiation of these two species of Fasciola.

Genotyping of Giardia lamblia isolates from human in southern Iran.

Giardia lamblia cysts isolated from human faeces in South of Iran were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. Among 205 faecal samples from microscopically diagnosed giardiasis patients, the gdh gene was amplified from 172 cases with a semi nested PCR assay and typed by RFLP analysis. Of the 172 positive samples, 128 (74.41%) were typed as assemblage AII, 30 (17.44%) assemblage BIII, 6 (3.49%) assemblage BIV and in 8 (4.66%) isolates, mixed assemblages AII and BIV were detected. Clinical features were available for 52 successfully typed cases and the possible correlation of Giardia assemblages and clinical symptoms was evaluated. Both assemblages caused similar illness, but assemblage AII was significantly more frequently associated with abdominal pain, nausea and vomiting. Since these isolates, A and B, are of human origin, anthroponotic transmission of Giardia can be suggested for the route of infection in this region.

In vitro cultivation of axenic amastigotes and the comparison of antioxidant enzymes at different stages of Leishmania tropica.

The present study aimed to establish a simple method to yield large amounts of Leishmania tropica amastigote-like forms in axenic cultures and to compare the superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes at different stages of L. tropica. Different culture conditions were tested to find the optimum condition of axenic amastigotes generation. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were determined at logarithmic and stationary phases and axenic amastigote stage of the parasite. A high proportion (88%) of amastigote-like forms of L. tropica was observed in BHI medium supplemented with 20% FCS, pH 4.5, and incubated at 37ºC with 5% CO(2). The results showed that SOD activity was at the lowest level in the logarithmic phase of promastigotes and increased towards the stationary phase of promastigotes and amastigote stage. The results showed that the optimum condition for differentiation of L. tropica promastigotes to axenic amastigotes was BHI medium containing 20% FCS at pH 4.5, incubated at 37ºC in the presence of 5% CO(2). It seems that SOD, but not GPX is a major determinant of intracellular survival of the parasite.

Characterization of the metacaspase 1 gene in Plasmodium vivax field isolates from southern Iran and Italian imported cases.

Plasmodium vivax is still the more prevalent human Plasmodium outside Africa and despite this fact, there is still a deep lack of knowledge on its biology. Metacaspases are cysteine proteases related to metazoan caspases, involved in programmed cell death. Here, we have characterized the P. vivax metacaspase 1 gene in a total of 63 vivax isolates, 32 isolates collected in southern Iran and 31 Italian imported isolates originating from 12 different endemic countries. We have firstly identified DNA size polymorphism in P. vivax metacaspase 1 gene. A total of four different allelic sizes were found, resulting from the insertion of 1 to 4 tandem repeat units located within the intronic region of the P. vivax metacaspase 1. Similarly, we also have identified four distinct allelic types by using vivax merozoite surface protein-1 size polymorphism analysis.

In vitro cultivation of axenic amastigotes and the comparison of antioxidant enzymes at different stages of Leishmania tropica

The present study aimed to establish a simple method to yield large amounts of Leishmania tropica amastigote-like forms in axenic cultures and to compare the superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes at different stages of L. tropica. Different culture conditions were tested to find the optimum condition of axenic amastigotes generation. Superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities were determined at logarithmic and stationary phases and axenic amastigote stage of the parasite. A high proportion (88%) of amastigote-like forms of L. tropica was observed in BHI medium supplemented with 20% FCS, pH 4.5, and incubated at 37ºC with 5% CO2. The results showed that SOD activity was at the lowest level in the logarithmic phase of promastigotes and increased towards the stationary phase of promastigotes and amastigote stage. The results showed that the optimum condition for differentiation of L. tropica promastigotes to axenic amastigotes was BHI medium containing 20% FCS at pH 4.5, incubated at 37ºC in the presence of 5% CO2. It seems that SOD, but not GPX is a major determinant of intracellular survival of the parasite.

A comparative study of antigen and antibody detection in visceral leishmaniasis using serum and urine-based ELISA.

An antigen-based ELISA system was evaluated for diagnosis of visceral leishmaniasis (VL). Urine samples from confirmed VL cases were tested by the system in comparison with urine samples from patients with non-VL infectious disease and patients with non-infectious diseases. Antigen was detected in urine of 21 out of 35 (60%) of VL cases. No cross reaction was found with samples from healthy individuals except in 3 samples from non-VL infectious diseases. Two samples from cutaneous leishmaniasis patient and one from patient with toxoplasmosis. The results obtained with the antigen-based ELISA were compared to those obtained with direct agglutination test (DAT), an antibody-based ELISA and indirect immunofluorescent antibody (IFA) revealed that the antigen-based ELISA is comparable in terms of specificity (91.2%; 95% CI=75.2-97.7%) but with a lower sensitivity (60%; 95% CI=42.2-75.6%). These results suggest that the antigen detection in urine by the noninvasive antigen-based ELISA system might offer a useful method for diagnosis of VL.

Asymptomatic human carriers of Leishmania infantum: possible reservoirs for Mediterranean visceral leishmaniasis in southern Iran.

Over the last decade, the incidence of visceral leishmaniasis (VL) has increased in many districts of the province of Fars, in southern Iran. Recent epidemiological reports indicate that asymptomatic human infections with Leishmania infantum (the causative agent of VL throughout the Mediterranean basin) occur more frequently in Iran than was previously believed. Between 2004 and 2006, blood samples were collected from 802 apparently healthy subjects from communities, in the north-west and south-east of Fars province, where VL cases had been recorded. Each of these samples was tested for anti-Leishmania antibodies, in direct agglutination tests (DAT), and for L. infantum kinetoplast DNA, in PCR-based assays. Of the 426 subjects from north-western Fars, eight (1.9%) were found seropositive and 68 (16.0%) PCR-positive. The corresponding values for the 376 subjects from south-eastern Fars were lower, with five (1.3%) seropositive and 32 (8.5%) PCR-positive. Of the 100 PCR-positive subjects, 18 (18.0%) each lived in a household in which there had been a case of VL, and six (6.0%) had had VL themselves (in each case, more than a year before the blood sampling for the present study). Although 21 of the PCR-positives have now been followed-up for at least 18 months, none has developed symptomatic VL. Since positivity in the PCR-based assay probably indicated the presence of L. infantum amastigotes in the peripheral blood of 12.5% of the subjects, it is clear that asymptomatic human carriers of L. infantum are quite common in the study areas and probably act as reservoirs in the transmission of the parasite, to humans and to dogs, by sandflies.

A search for the rodent hosts of Leishmania major in the Larestan region of southern Iran: demonstration of the parasite in Tatera indica and Gerbillus sp., by microscopy, culture and PCR.

Zoonotic cutaneous leishmaniasis (CL) caused by Leishmania major occurs widely in Iran, where several species of rodent serve as the parasite's 'reservoir' hosts. In an attempt to identify the rodent hosts in the Larestan region, which lies in the Fars province of southern Iran (where the incidence of human CL has been rising), 32 rodents (20 Tatera indica, eight Meriones crassus, four Gerbillus sp.) were caught and checked for leishmanial infection. Using two detection methods (the microscopical examination of stained tissue smears and the culture of tissue samples) and a PCR to identify any leishmanial parasites detected, L. major was identified in six of the rodents caught: two male T. indica from Alamarvdasht, two female T. indica from Lamerd, and two females of the genus Gerbillus (one caught in Lamerd and one in Lar). Although the samples were too small to prove that M. crassus is not a significant host of L. major in Larestan, they were large enough to indicate that T. indica and members of the genus Gerbillus serve as reservoir hosts of L. major in the region. Tatera indica appears to be an important host of L. major across much of Iran but this appears to be the first time that the genus Gerbillus has been found to be involved in the epidemiology of CL in the country.

Characterization of Leishmania isolated from unhealed lesions caused by leishmanization.

Leishmania parasites were isolated after 13 and 8 years from the unhealed lesions of 2 soldiers who had been immunized against leishmaniasis during the war between Iraq and the Islamic Republic of Iran. Isoenzyme characterization on these isolates using 11 enzyme systems was carried out and the results were compared with the enzyme profiles of the original isolates of L. major used for leishmanization. Minor enzymatic differences in glucose-6-phosphate dehyrognase and phosphoglucomutase were observed but otherwise the strains appeared unchanged.

Characterization of Leishmania isolated from unhealed lesions caused by leishmanization

Leishmania parasites were isolated after 13 and 8 years from the unhealed lesions of 2 soldiers who had been immunized against leishmaniasis during the war between Iraq and the Islamic Republic of Iran. Isoenzyme characterization on these isolates using 11 enzyme systems was carried out and the results were compared with the enzyme profiles of the original isolates of L. major used for leishmanization. Minor enzymatic differences in glucose-6-phosphate dehyrognase and phosphoglucomutase were observed but otherwise the strains appeared unchanged

Characterization of Leishmania isolated in Iran: 1. Serotyping with species specific monoclonal antibodies.

A total of 156 isolates of Leishmania from patients with cutaneous leishmaniasis and one isolate from gerbil were characterized using standard monoclonal antibodies (mAb) in ELISA and IFA test systems. The geographic distribution of the isolates was 30 isolates in Shiraz, 28 in Kerman and 98 in Tehran. A total of 63, 72, and three Leishmania promastigote isolates preferentially reacted with anti-Leishmania tropica mAb (A11), anti-Leishmania major mAb (T1) and anti-Leishmania infantum mAb (D2), respectively. Of the three isolates which reacted with anti-L. infantum mAb, two were dermotropic strains of L. infantum and one was the organism isolated from gerbil. Mixed reactions with the monoclonal antibodies were also observed as six isolates reacted with both anti-L. tropica and anti-L. major mAbs, five reacted with anti-L. tropica and anti-L. infantum mAbs and two with anti-L. major and anti-L. infantum mAbs. The monoclonal antibodies used did not react with five of the isolates. The present investigation has identified the organisms from the known and newly emerged foci of cutaneous leishmaniasis in Iran.