A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran.

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per µL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per µL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.

Epidemiological features of cutaneous leishmaniasis and distribution of sand flies in an endemic area in southeast of Iran.

INTRODUCTION: Cutaneous leishmaniasis (CL) is a widespread tropical infectious disease in the world. It is one of the most important health problem in Iran which is endemic in different parts of country. This study was conducted to determine epidemiological features of CL and distribution of sand flies in Hajiabad County, one of the important CL foci in southeast of Iran. METHODS: This descriptive cross-sectional study was conducted from March 2019 to March 2020.All of the suspected CL cases with skin lesions who referred to Hajiabad County health centers and all actively detected patients were clinically and parasitologically examined for CL. Demographic and clinical data of all patients were recorded. Moreover, in this study, sand flies were collected monthly from four typical plain and foothill villages during their active season (May-November) using sticky trap. Sand flies were mounted as permanent microscopic slides, using Puri's medium, and identified by taxonomic keys. Data were analyzed using SPSS.21 software and descriptive statistics. RESULTS AND DISCUSSION: A total of 70 confirmed cases of CL were recorded; the incidence rate of the disease was 101 per 100,000 people. The most infected age group was 0-10 years, with a rate of 64.3%. Males were infected more than females and the majority of cases (85.7%) were recorded from rural areas. Most of the cases had 1 lesion (51.4%) and the most lesions (55.8%) were in upper extremities. During the study period, 832 sand fly specimens comprised of ten species of Sergentomyia and seven of Phlebotomus were collected and identified. The most prevalent species was P. papatasi (47.12%), followed by P. alexandri (8.41%) and P. salehi (6.25%). Among the collected Phlebotomus species, P. papatasi and P. sergenti are known as the primary vectors of CL and P. alexandri, P. salehi, and P. caucasicus, play the main role as the secondary vectors of CL in Iran. CONCLUSION: This study has revealed that CL is endemic in Hajiabad County and there are five CL vectors that are distributed in this County and some of them are more prevalent in plain areas. These findings can be used as a basis for implementation of interventions toward vector control, which may help in suppression of vector density, and consequently, control of CL in the study area.

Tadalafil Reversed H-89 - and Scopolamine - Induced Spatial Learning Impairments in Male Rats.

Accumulated evidence shows that the cAMP and cGMP signaling pathway plays an important role in memory function and neuronal plasticity. Phosphodiesterase 5 (PDE5) is a hopeful therapeutic target in AD (Alzheimer disease), and PDE5 inhibition may be a good therapeutic strategy for the treatment of AD. In the present study, the four-day bilateral intra-hippocampal infusion of H-89 as a protein kinase AII inhibitor (10 µM/side) and intra-peritoneal injections of tadalafil (20 mg/kg) and scopolamine (0.5 mg/kg) alone and also on combination on spatial learning in Morris water maze (MWM) were investigated. DMSO and saline were used as controls for H-89 and other mentioned drugs, respectively. Rats were trained for 4 days; each day included one block of four trials. Post- training probe trial tests were performed on day 5. Administration of H-89 and scopolamine led to a significant impairment in spatial learning compared to their related controls. But, combination of tadalafil/H-89 or tadalafil/scopolamine reversed H-89 or scopolamine- induced spatial learning deficits in MWM. Taken together, these results showed the probable regulatory effects of cGMP on cholinergic and cAMP/PKA signaling pathways in co-administrations of these mentioned drugs on spatial learning in MWM.

Modulation of Hypertrophic Scar Formation Using Amniotic Membrane/Electrospun Silk Fibroin Bilayer Membrane in a Rabbit Ear Model.

Hypertrophic scarring is a dermal disorder resulting from collagen and other extra cellular matrix protein depositions following the deep trauma, severe burn injury, and surgery incisions. A variety of therapeutic procedures are currently available, however, achieving an ideal treatment method remains a challenge. In our recently published report, a 3D bilayered decellularized human amniotic membrane/electrospun silk fibroin membrane was fabricated and characterized for regenerative medical applications. To obtain a solid bind between two layers, the samples were immersed in 70% ethanol. In this study, the effects of amniotic membrane/electrospun silk fibroin on minimizing the postinjury hypertrophic scar formation were determined in the rabbit ear model. In vivo experiments were carried out to assess the bilayer membrane characteristics on full thickness hypertrophic scar at days 28 and 50 postimplantations. A significant decrease in collagen deposition and expression and increased expression and deposition of MMP1 in the wound bed were observed on the wounds dressed with bilayered membrane when compared to the amniotic membrane alone and controls (wound with no implant). The current study shows that our fabricated construct has potential as an efficient antiscarring wound dressing material and may also serve for the subsequent soft tissue engineering needs.

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins.

Cysteine PEGylation includes several steps, and is difficult to manage in practice. In the current investigation, the cysteine PEGylation of erythropoietin analogs was examined using computational and nonglycosylated systems to define a simpler approach for specific PEGylation. Two model analogs (E31C and E89C) were selected for PEGylation based on lowest structural deviation from the native form, accessibility, and nucleophilicity of the free thiol group. The selected analogs were cloned and the expression was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using Coomassie blue staining and anti-His monoclonal antibody, respectively. PEGylation with 20 kDa mPEG-maleimide resulted in 79% and 82% conjugation yield for E31C and E89C nonglycosylated erythropoietin (ngEPO) analogs, respectively. The size distribution and charge analysis showed an increase in size and negative charge of the PEGylated forms compared with nonconjugated ones. Biological assay revealed that E31C and E89C mutations and subsequent PEGylation of ngEPO analogs have no deleterious effects on in vitro biological activity when compared to CHO-derived recombinant human erythropoietin. In addition, PEG-conjugated ngEPOs showed a significant increase in plasma half-lives after injection into rats when compared to nonconjugated ones. The development of the cysteine-PEGylated proteins using nonglycosylated expression system and in silico technique can be considered an efficient approach in terms of optimization of PEGylation parameters, time, and cost.

Direct analysis of human breath ammonia using corona discharge ion mobility spectrometry.

In this study, ammonia in human breath was directly determined using corona discharge ionization ion mobility spectrometry (CD-IMS) technique with several important advantages including high sensitivity, low cost, high speed, and ease of maintenance. The temperature effect on the ammonia signal was evaluated too. The results indicated that the best temperature for the investigation of breath ammonia was 150°C. The analytical results showed that the linear dynamic range was between 12 and 810ppb and the detection limit was 6.6ppb. The relative standard deviation (RSD) was obtained to be 5, 3, and 3 for 290, 348, and 522ppb, respectively. The amounts of ammonia in breath of eight healthy volunteers were measured. The values were between 236 and 1218ppb. Also, the inequality in breath ammonia levels was scrutinized over a 6h working day for three healthy volunteers. The results showed a drop in breath ammonia from the morning amount to the mid-day measurement and then, a progressive increase while the day continued. In addition, the amounts of ammonia were determined to be 1494-1553ppb in exhaled breath of two renal failure patients. The results obtained in this work revealed that the method was conveniently established without any considerable sample pretreatment for direct analysis of ammonia in human breath.