Automated Camera Lucida Method with Colored Images through Integration of Hardware and Software in Microscopic Zooming.

Automating the camera Lucida method which is a standard way for focusing microscopic images is a very challenging study for many scientists. Hence, actually combining hardware and software to automate microscopic imaging systems is one of the most important issues in the field of medicine as well. This idea reduces scanning time and increases the accuracy of user's results in this field. Closed-loop control system has been designed and implemented in the hardware part to move the stage in predefined limits of 15°. This system produces 50 consecutive images from parasites at the mentioned spatial distances in two directions of the z-axis. Then, by introducing our proposed relational software with combining images, a high-contrast image can be presented. This colored image is focused on many subparts of the sample even with different ruggedness. After implementing the closed-loop controller, stages movement was repeated eight times with an average step displacement of 20 µm which were measured in two directions of the z-axis by a digital micrometer. On average, the movement's error was 1 µm. In software, the edge intensity energy index has been calculated for image quality evaluation. The standard camera Lucida method has been simulated with acceptable results based on experts' opinions and also mean squared error parameters. Mechanical movement in stage has an accuracy of about 95% which will meet the expectations of laboratory user. Although output-focused colored images from our combining software can be replaced by the traditional fully accepted Camera Lucida method.

Molecular and microscopy detection of Pneumocystis jirovecii in hospitalized patients during the COVID-19 pandemic.

Introduction: Early detection of Pneumocystis jirovecii as an opportunistic pathogen that may endanger predisposed persons, including COVID-19 patients, may help to choose the optimal management. Methods: In this study, 585, including 530 COVID-19 patients, with clinical and radiological evidence of respiratory diseases, were investigated for P. jirovecii screening. Clinical specimens were examined by direct microscopy and PCR, and randomly selected positive PCR products were confirmed through DNA sequence analysis. Results: Thirty-one (5.3%) samples were positive in P. jirovecii-specific nested-PCR, while by direct microscopic tests, Pneumocystis was observed in 22 (3.76%) samples. Males (61.7%) and patients over 50 years old (75.6%) were more commonly affected than others, and malaise and fatigue (84%), and wheezing (75%) were the most common symptoms, followed by fever (40.48%) and dyspnea (39.51%). Among the Pneumocystis-positive patients, three cases had coinfection with Aspergillus fumigatus, A. flavus, and A. niger (each n = 1), as documented by direct microscopy, culture, and species identification by PCR-sequencing. Conclusion: Pneumocystis pneumonia is still a diagnostic challenge; therefore, additional large-scale studies are needed to clarify the epidemiology of the disease in immunocompromised or COVID-19 patients.

Identification and Subtyping of Cryptosporidium parvum and Cryptosporidium hominis in Cancer Patients, Isfahan Province, Central Iran.

Background: Cryptosporidium spp. are protozoan parasites that cause diarrhea in humans and animals. Subtyping data about Cryptosporidium spp. in Isfahan, Iran is limited; therefore, we aimed to study the prevalence rate of Cryptosporidium spp. in cancer patients, associated risk factors, and subtypes of Cryptosporidium spp. Methods: Fecal samples were collected from 187 cancer patients from the Oncology Department of Seyed-al-Shohada Hospital, Isfahan University of Medical Sciences during 2014-2020 and screened for Cryptosporidium spp. using microscopical techniques. Nested PCR amplifying 18S rRNA gene was used to detect Cryptosporidium spp. in samples, followed by subtyping using nested PCR amplifying gp60 sequences. Results: Overall, the rate of infection with Cryptosporidium spp. was 4.3% (n=8). Five samples out of eight samples were identified as Cryptosporidium spp. using a nested PCR for the 18S rRNA gene, two subtypes of C. parvum named IIaA18G3R1 (n = 2) and IIaA17G2R1 (n = 2), and one subtype of C. hominis named IbA6G3 were identified by sequencing of the gp60. The IbA6G3 subtype has rarely been detected in other investigations. Conclusion: This is the first survey on the subtyping of Cryptosporidium spp. in this region. The results of the present survey show both zoonotic and anthroponotic transmission routes in the region.

Prevalence of Intestinal Protozoan Infection in Patients with Ulcerative Colitis (UC) in Isfahan, Iran.

BACKGROUND: Determination of the prevalence of intestinal protozoan infection is a fundamental step to set up an effective control program to improve the health status of society and to establish efficient strategies. Intestinal pathogen and even non-pathogen protozoa consider as major causes of disease in patients with gastrointestinal problems. The objective of this study is to determine the prevalence of intestinal protozoan infection in patients with ulcerative colitis (UC) in Isfahan, Iran. METHODS: The descriptive cross-sectional study carried out from 2013 to 2018 in Isfahan, Iran. One thousand nine hundred and sixty-five samples of feces from patients with UC collected and each sample examined using direct wet mounting with normal saline and iodine and sedimentation tests such as formol-ethyl acetate concentration and trichrome-staining methods. RESULTS: From 655 patients, 185 (28.2%) infected with Giardia lamblia followed by Blastocystis hominis (27.3%), Endolimax nana (14.4%), Entamoeba coli (11.5%), Iodamoba butschlii (4.7%), Entamoeba histolytica (1.4%), and Chilomastix mesnili (0.6%). CONCLUSIONS: This study revealed a high prevalence of infection with at least one or six non-pathogenic and pathogenic intestinal protozoa in UC patients in the Isfahan region. Intestinal protozoa are a challenging public health problem wherever health care is limited in the area. The emergence of UC in the world results in the need to study etiologic factors. In order to obtain further information about the etiology of disease, we investigated the prevalence of intestinal protozoan infection in patients with UC in Isfahan, Iran.

Automated Drawing Tube (Camera Lucida) Method in Light Microscopy Images Analysis Can Comes True.

BACKGROUND: In a light microscope, image acquisition with different component depths is difficult, and there are various approaches for solving this problem. One of the common approaches is Camera Lucida (CL). This method has some disadvantages such as time-consuming, handed problems in painting, causing user boring, and produce gray scale output images. AIMS AND OBJECTIVES: In this study, we purposed a novel-combined hardware and software method. In this article, we try to present an automated method for our designed microscope. MATERIALS AND METHODS: We have done a project with designed code number 377,694 to design and implement an upgraded light microscope. That project was about automatic movement of a stage with closed-looped control of a servomotor. Furthermore, automated camera catches images in predefined positions. That project has acceptable results in different parts, which encourage us to work on this study. This study help specialist have good fixative of all components in a sample. It is about trying to have useful Lucida Camera (drawing tube) in an automated scheme. RESULTS: This method is an acceptable usual way for microscopic specialists, but with some disadvantages. It is time-consuming and boring that effect on the accuracy of results. Hence, how can be good if automated similar method could be implemented is exciting and affective. This studies idea comes from the basis of manual drawing tube (CL) method. In this experimental study, we have taken 400 handed an image of microorganisms. Captured images are from its whole body or various organs. They have been captured in different z-axis positions of stage, and hence components with different depths could be focused. Each patch checked for its edge strength to choose highest resolutions sub image and reconstruct focused image like a puzzle. This process has been continued for all areas to merge and complete reconstructed image as output. CONCLUSION: Comparing edge strength with other images and mean square error with manual focused on confirm our method with pleasure outcomes. Furthermore, independent focusing of an internal component in a sample body has been surveyed. It helps to have better resolution in internal selected component for more analysis and replace in its primitive image. This article presents efficient consequences with good accuracy and saving time in process period, which could be useful in different microscopes types and various samples type.

Synthesis and In vitro Leishmanicidal Activities of Six Quercetin Derivatives.

BACKGROUND: Today, leishmaniasis is a widespread, infectious parasitic disease caused by Leishmania spp. Natural-derived compounds are likely to provide a valuable source of new pharmaceuticals, and among them, quercetin derivatives may have antileishmanial effects. The antileishmanial activity of 3,5,7,3',4'-pentahydroxyflavonol (quercetin) derivatives is partly attributed to the position and pKa of phenolic or catechol hydroxyl groups. Therefore, to optimize their leishmanicidal effect, the structural features of quercetin and its derivatives were improved by acylation or alkylation of hydroxyl groups and changing their pKa and consequently their activities. MATERIALS AND METHODS: In this study, during a regioselective method, quercetin derivatives were synthesized. The structures of synthesized compounds were confirmed by mass, IR, 1H-, and 13C-NMR spectral data. The antileishmanial activities of compounds 1-6 were compared with glucantime as the standard drug against promastigotes of Leishmania major using standard cell-based leishmanicidal assay. RESULTS: In this study, during a regioselective method, two 7-O-quercetin derivatives (5 and 6), and three quercetin acetate derivatives (2, 3, and 4) were synthesized. In detail, the IC50 values found against L. major were (1) 2.5 ± 0.92; (2) 2.85 ± 0.99; (3) 15.5 ± 1.95; (4) 13.5 ± 3.5; (5) 2.6 ± 0.57; and (6) 1.3 ± 0.35 µM while IC50 value of glucantime as the standard drug was 88.5 ± 9.47 µM. CONCLUSIONS: The present study showed an effective antileishmanial activity of quercetin semisynthetic compounds (1-6) against in vitro promastigotes of L. major. Among them, quercetin analogs with more lipophilic and iron-chelating activity showed more antiparasite activity.

Evaluation of the Antimalarial Effect of Ferulago angulata (Schlecht.) Boiss. Extract and Suberosin Epoxide Against Plasmodium berghei in Comparison with Chloroquine Using in-vivo Test.

Resistance to most antimalarial drugs has encouraged the development of novel drugs. An alternative source for discovering such drugs is natural products. Some Ferulago species are used in folk medicine for their sedative, tonic and anti-parasitic effects. Besides, coumarins isolated from this genus found to have in vitro anti-leishmanicidal effect. The present study is aimed to evaluate the in-vivo antimalarial activity of Ferulago angulata (Schlecht.) Boiss. extract and suberosin epoxide, using suarian mice. A rodent malaria parasite, Plasmodium berghei was used to inoculate healthy male Swiss Albino mice of age 6-8 weeks and weight 23-27 g. Hydro-alcoholic extract of F. angulata (20, 100, 300, 600 mg/Kg) and suberosin epoxide suspension (10, 30, 50, 100 mg/Kg) were administered subcutaneously. Parameters including percentage of parasitemia, suppression of parasitemia and mean survival time were determined using standard test such as peterÙ¬s. Chemo-protective effects were exerted by the crude extract and suberosin epoxide. Maximum effect was observed with the larger doses of the crude extract and suberosin epoxide. Suberosin epoxide increased the survival time compared to chloroquine. However, the results of this study indicate that the plant has a promising anti-plasmodial activity against plasmodium berghei. Thus, it could be considered as a potential source of new antimalarial agents. Suberosin epoxide at the dose of 100 mg/Kg possesses relatively significant antimalarial effect. Chemical derivatization of the parent compound or preparation of the modified formulation is required to improve its systemic bioavailability.

Effect of two hydatid cyst antigens on the growth of melanoma cancer in C57/black mice.

Hydatid cyst is the larval stage of Echinococcus granulosus. In previous studies inhibitory effect of this parasite on cancer cell growth in culture medium has been shown. In this study effect of hydatid cyst antigens on tumor growth in experimental animals has been investigated. Two antigens of hydatid cyst including protoscolices excretory secretory antigen and hydatid fluid absorbed on alum as adjuvant were injected to two groups of C57/black mice as case groups. Control groups were injected with only saline and alum. All mice then were injected with melanoma cells. Both antigens reduced the tumor size in mice in case groups. The difference of tumor size in mice in case groups and control group was statistically significant. In conclusion, anti-tumor effect of hydatid cyst antigens may be related to antigenic similarities which exist between hydatid cyst and cancer cells.

Manipulation of IL-10 gene expression by Toxoplasma gondii and its products.

BACKGROUND: This study was designed to evaluate whether or not T. gondii and its derivatives can change the gene expression level of IL-10 in murine leukocytes in vivo. METHODS: Fifty BALB/c mice were divided into 5 groups, four of which received the excretory/secretory product (ESP) from cell culture medium, the ESP from cell free medium, the Toxoplasma lysate product (TLP) and the active tachyzoites, respectively. The fifth group was considered as control and received PBS. The peritoneal leukocytes from the mice were collected. Their total RNA were extracted and converted to cDNA and the gene expression levels of IL-10 in the samples were evaluated by quantitative real time-PCR using the REST-2009 software. RESULTS: The findings showed a decrease in the expression level of IL-10 in the TLP group (p=0.004). Moreover, the IL-10 gene expression level was upregulated in the group of the ESP from cell culture medium (p=0.04) and the active tachyzoite group (p=0.04). The expression of IL-10 gene in the group of ESP from cell-free medium was not significant compared to the control one (p=0.45). CONCLUSION: T. gondii and its derivatives are able to increase (the active T. gondii tachyzoite and the ESP from cell culture medium) and decrease (the TLP) the gene expression level of IL-10 in a murine model. The question remains to be examined in further study about which molecules are involved in this process.

The gene expression level of IFN-γR1 and IFN-γR2 in a murine model treated with Toxoplasma gondii and its products.

AIM: To evaluate the effect of active T. gondii tachyzoites and its products on the gene expression level of IFN-γR1 and IFN-γR2 in a murine model. BACKGROUND: Many studies have shown that the parasite Toxoplasma gondii utilizes different mechanisms to inhibit the function of IFN-γ, but the parasite effect on the function of IFN-γR1 and IFN-γR2 is still unclear. PATIENTS AND METHODS: Toxoplasma lysate product (TLP), excretory/secretory products (ESPs) obtained from cell free and cell culture media as well as active tachyzoites were injected separately to their respective group each consisted of 10 BALB/c mice. One control group of 10 mice received phosphate buffered saline (PBS). All of the mice were euthanized three days after the last injection and then their peritoneal leukocytes were harvested separately. The total RNA was extracted from the samples, converted to cDNA, and the gene expression level of IFN-γR1 and IFN-γR2 was assessed in all of the treated groups relative to the control one. RESULTS: There was no significant difference between each of the treated groups relative to the control group concerning the gene expression level of IFN-γR2 (P> 0.05). Furthermore, the gene expression level of IFN-γR1 in two groups of TLP (P= 0.04) and ESP obtained from cell free medium (P= 0.008) showed a significant difference relative to the control group. CONCLUSION: Findings of this study revealed a new aspect of host-T. gondii interaction in that this parasite is able to downregulate IFN-γR1 to reduce the IFN-γ effects on the infected cell.

First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis.

AIM: The present study was aimed to evaluate E. granulosus genotypes isolated from goats using HRM analysis in Isfahan province. BACKGROUND: Cystic echincoccosis, so-called hydatidosis, is widespread infection caused by the larval stage of Echinococcus granulosus. This is an important zoonotic disease worldwide, especially in the developing countries such as Iran. To date, molecular studies mainly based on the mitochondrial DNA sequences have identified distinct genotypes termed G1-G10 which can differ in some characteristics such as the growth and infectivity to different intermediate hosts or the survival rate in the definitive hosts that are important for the development of control strategies. METHODS: From August to December 2014, 1341 goats were investigated and hydatid cysts were collected from the liver and lungs of 43 infected goats in Isfahan province abattoirs, Isfahan, Iran. Total genomic DNA was extracted from each sample, amplified for the presence of polymorphism of mitochondrial gene coding for cytochrome c oxidase subunit 1 (CO1), using high resolution melting curve (HRM) method. RESULTS: the results of HRM analysis using the sequence of CO1 gene for 43 Echinococcus granulosus isolates from goats showed 31, 2 and 10 isolates were identified as G1, G2, and G3 genotypes, respectively. CONCLUSION: G1 is the predominant genotype in the isolated goat samples in Isfahan province, and the presence of G2 strain was reported for the first time in goat in Iran.

Genetic diversity of Echinococcus granulosus in center of Iran.

Hydatid cyst caused by Echinococcus granulosus is one of the most important parasitic diseases around the world and many countries in Asia, including Iran, are involved with this infection. This disease can cause high mortality in humans as well as economic losses in livestock. To date, several molecular methods have been used to determine the genetic diversity of E. granulosus. So far, identification of E. granulosus using real-time PCR fluorescence-based quantitative assays has not been studied worldwide, also in Iran. Therefore, the aim of this study was to investigate the genetic diversity of E. granulosus from center of Iran using real-time PCR method. A total of 71 hydatid cysts were collected from infected sheep, goat, and cattle slaughtered in Isfahan, Iran during 2013. DNA was extracted from protoscolices and/or germinal layers from each individual cyst and used as template to amplify the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) (420 bp). Five cattle isolates out of 71 isolates were sterile and excluded from further investigation. Overall, of 66 isolates, partial sequences of the cox1 gene of E. granulosus indicated the presence of genotypes G1 in 49 isolates (74.2%), G3 in 15 isolates (22.7%), and G6 in 2 isolates (3.0%) in infected intermediate hosts. Sixteen sequences of G1 genotype had microgenetic variants, and they were compared to the original sequence of cox1. However, isolates identified as G3 and G6 genotypes were completely consistent with original sequences. G1 genotype in livestock was the dominant genotype in Isfahan region, Iran.

Molecular identification of Giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population?

AIM: The aim of this study is to investigate the molecular identification of Giardia lamblia in patients with diarrhea. BACKGROUND: Giardiasis caused by Giardia lamblia is a common intestinal disease. Although this parasitic infection found in mammals including human, pets and livestock, but few species within the genus Giardia can infects humans. G. lamblia have seven complex genotypes termed (A-H). Genotype A and B the main causes of human infections. PATIENTS AND METHODS: Sixty seven microscopically positive G. Lamblia samples were collected from clinical laboratories in Isfahan province between June 2013 and February 2014. Extraction of genomic DNA was performed for 65 concentrated cysts and 2 cultured trophozoites. Partial sequences of tpi including 148-bp and 81-bp were amplified for detection the genotypes A and B using RFLP- PCR protocol respectively. RESULTS: PCR results showed that out of 67 patients with giardiasis infection, genotype A (148 bp) was detected in 40 isolates (59.70%) compared to genotype B (81 bp) isolated was detected in 25 isolates (37.31%). Also two isolates (2.98%) had mix infection infected with genotype A and B. By comparing the frequency of genotype A (81.8%) and genotype B (13.6%), we found that genotype A is six times higher prevalence than genotype B in patients with diarrhea. CONCLUSION: We suggest that using sensitive techniques and larger sample for detection of G. lamblia genotypes and their subtypes would be necessary for investigation the immune system respond and correlation with diarrhea in the future studies in Iran.

Isolation of Cryptococcus neoformans and other opportunistic fungi from pigeon droppings.

BACKGROUND: Invasive fungal infections cause considerable morbidity and mortality in immunocompromised hosts. Pigeon droppings could especially be a potential carrier in the spread of pathogenic yeasts and mold fungi into the environment. The objective of this study was to isolation of Cryptococcus neoformans and other opportunistic fungi from pigeon droppings. MATERIALS AND METHODS: One hundred twenty samples of pigeon droppings were suspended 1:10 in saline solution and then cultured. Identification of C. neoformans was performed on bird seed agar, presence of a capsule on India ink preparation, urease production on urea agar medium and RapID yeast plus system. The identification of candida species was based on micro-morphological analysis on corn meal-Tween 80 agar, RapID yeast plus system and growth in CHROMagar candida. The identification of other fungi was based on macromorphologic, microscopic, biochemical and physiological characteristics. RESULTS: The highest frequency of yeasts and mold fungi were observed in Candida albicans 6.6% and Penicillium spp. 25%. The frequency rate of C. neoformans isolation was 2.5%. CONCLUSION: Several types of fungi are present in pigeon droppings that can spread in environment and transmit to children and elderly as well as immunocompromised patients who are at increased risk of contracting opportunistic diseases.

The Correlation between Intestinal Parasitic Infections and Bruxism among 3-6 Year-Old Children in Isfahan.

BACKGROUND: Given the fact that bruxism is a prevalent oral habit among children and a potential destructor of oral tissues, the present study aimed to investigate the relationship between intestinal parasitic infections and bruxism among kindergarten children. METHODS: Questionnaires were administered among parents of kindergarten children in Isfahan to select 50 children identified by their parents to have the habit of bruxism and 50 without the habit as control group. Informed consent was obtained prior to the investigation. Parents were delivered sampling instruments with proper instructions to collect stool samples from both groups for parasitological tests. The diagnostic parasitological tests involved the direct stool smear, formol-ether concentration, and Scotch tape tests. Comparison for the frequency distribution of intestinal parasitic infections between the two groups was performed using the chi-square test (α = 0.05). RESULTS: Parasitic infections were observed in 19 (11/50 cases and 8/50 controls) children. A statistically significant relationship was observed between infection with pathogenic parasites and bruxism (P < 0.05). CONCLUSION: Our findings suggest that pathogenic parasites may serve as the cause of initiation of bruxism habits among children.