Metagenomic analysis of the ocular toxoplasmosis in children uveitis from Fayoum governorate, Egypt.

Granulomatous anterior uveitis with single or numerous gelatinous nodules was found in children living in rural Egypt. All ocular diseases were originally thought to be water-born and related to digenic flukes. The current study sought to learn more about the causes of anterior granulomatous uveitis in Egyptian youngsters who used to swim in rural water canals. 50 children with eye lesions that had not responded to medical treatment were recruited. Four samples were surgically extracted and examined using real-time PCR, transmission electron microscopy (TEM), and shotgun metagenomic sequencing (SMS). Toxoplasma gondii was detected free within the syncytium's distal section, while the proximal part exhibited active synthesis of a presumably extra-polymeric material, possibly released by the microbial population. Toxoplasma gondii was found in 30 samples. Serologically, distinct anti-Toxoplasma antibodies were not found in 91.6% of patients. SMS showed that the T. gondii ME 49 strain had the greatest percentage (29-25%) in all samples within an Acinetobacter-containing microbial community. These findings suggested that these bacteria entered the body via the exterior route rather than the circulatory route. The lack of genetic evidence for subsequent parasite stages invalidates the prior findings about the assumed trematode stage.

Impact of atorvastatin and mesenchymal stem cells combined with ivermectin on murine trichinellosis.

Trichinellosis is one of the global food-borne parasitic diseases that can cause severe tissue damage. The traditionally used drugs for the treatment of trichinellosis have limited efficacy against the encysted larvae in the muscular phase of the disease. Therefore, this study aimed to evaluate the role of atorvastatin and mesenchymal stem cells combined with ivermectin against different phases of Trichinella in experimentally infected mice. A total of 120 male Swiss albino mice were divided into two major groups (n = 60 of each), intestinal and muscular phases. Then, each group was subdivided into 10 subgroups (n = 6); non-infected control, infected non-treated control, infected ivermectin treated, infected atorvastatin treated, infected mesenchymal stem cells treated, infected combined ivermectin and atorvastatin treated, infected combined mesenchymal stem cells and ivermectin treated, infected combined mesenchymal stem cells and atorvastatin treated, infected combined mesenchymal stem cells and a full dose of (ivermectin and atorvastatin) treated, and infected combined mesenchymal stem cells and half dose of (ivermectin and atorvastatin) treated. Mice were sacrificed at days 5 and 35 post-infection for the intestinal and muscular phases, respectively. The assessment was performed through many parameters, including counting the adult intestinal worms and muscular encysted larvae, besides histopathological examination of the underlying tissues. Moreover, a biochemical assay for the inflammatory and oxidative stress marker levels was conducted. In addition, levels of immunohistochemical CD31 and VEGF gene expression as markers of angiogenesis during the muscular phase were investigated. The combined mesenchymal stem cells and atorvastatin added to ivermectin showed the highest significant reduction in adult worms and encysted larvae counts, the most noticeable improvement of the histopathological changes, the most potent anti-inflammatory (lowest level of IL-17) and anti-angiogenic (lowest expression of CD31 and VEGF) activities, and also revealed the highly effective one to relieve the oxidative stress (lowest level of SOD, GSH, and lipid peroxidase enzymes). These observed outcomes indicate that adding mesenchymal stem cells and atorvastatin to ivermectin synergistically potentiates its therapeutic efficacy and provides a promising candidate against trichinellosis.

Toxoplasmosis and cytomegalovirus infection and their role in Egyptian autistic children.

Autism is a neurodevelopmental disorder with a significantly increased incidence rate across the world over the past few years. Toxoplasmosis and cytomegalovirus (CMV) infection are globally prevalent and have been associated with diverse neurological and psychiatric disorders. A few studies have demonstrated the role of toxoplasmosis and CMV as potential etiological factors for autism. Accordingly, this study was performed to estimate the relationship between toxoplasmosis and CMV infection in children with autism as well as to assess their impact on the Childhood Autism Rating Scale (CARS) score. A total of 45 autistic children (6 girls, 39 boys) and 45 (21 girls, 24 boys) healthy control children were enrolled in our study. Their blood samples were collected and tested for the presence of Toxoplasma and CMV (IgG and IgM) antibodies and DNA by ELISA and real-time PCR (RT-PCR), respectively. Toxoplasmosis was detected in 11 (24.4%) autistic children through the ELISA [10 (22.2%) IgG + /IgM - and 1 (2.2%) IgG + /IgM +]; however, RT-PCR assay recorded only 1 positive case (2.2%), while it was detected in 10 (22.2%) control children through ELISA [9 (20%) IgG + /IgM - and 1 (2.2%) IgG + /IgM +] and 1 (2.2%) by RT-PCR. On the other hand, CMV infection was detected in all autistic children with 44 (97.8%) testing positive by ELISA [24 (53.3%) IgG + /IgM - , 18 (40%) IgG + /IgM + and 2 (4.4%) IgG - /IgM +] and 25 (55.6%) testing positive by RT-PCR assay. In addition, ELISA assay recorded 43 (95.6%) [19 (42.2%) IgG + /IgM + and 22 (48.9%) IgG + /IgM - and 2 (4.4%) IgG-/IgM +] and RT-PCR recorded 21 (46.7%) positive samples in control children with CMV. No significant difference was noted between autistic and control children for the overall prevalence of Toxoplasma or/and CMV infection. Similarly, the CARS score indicated a non-significant difference with Toxoplasma or/and CMV infection. Our data does not show an association between autism and toxoplasmosis or/and CMV infection. Nevertheless, considering that autistic children are at a high risk of contracting these infections, further studies with a larger sample size are recommended.

Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats.

PURPOSE: The relationship between the genetic diversity of Blastocystis and immune surveillance in precancerous colons with blastocystosis is still under investigation. This study aimed to identify the genetic Blastocystis variants among 54 symptomatic human isolates and their relationship to mucosal immune surveillance in the precancerous polyps of experimentally infected rats. METHODS: Polymerase chain reaction and high-resolution melting (PCR/HRM) curves discriminated human symptomatic Blastocystis isolates into subtypes (STs)/intrasubtypes, which were orally administered to rats to induce experimental infection. Then, the mucosal immune responses of the infected colons were evaluated in relation to polyp formation through immunostaining to identify mucus MUC2 and determine mucosal immune cell (goblet, lymphocyte and mast) counts, secretory IgA levels and parasitic intestinal invasion. RESULTS: ST1, ST3, and ST4 were found in 18.5% (10/54), 54.7% (29/54), and 27.8% (15/54) of the samples, respectively. Then, the HRM curve discriminated ST3 into the wild, mutant, and heterozygous [17/54 (31.5%), 5/54 (9.3%), and 7/54 (12.9%)] intrasubtypes. ST1 and ST4 had no genetic variations. Precancerous polyps were detected in the colons of 40.5% of the infected rats. ST1 constituted 14.7% of these cases, while the wild, mutant, and heterozygous intrasubtypes of ST3 showed polyps in 12.9%, 5.5%, and 5.5% of cases, respectively. Only 1.9% of the polyps were related to ST4. MUC2 showed weak immunostaining in 44.5% of the infected colons, and 38.9% were polyp inducers. Low goblet cell numbers and high interepithelial lymphocyte counts were significantly associated with polyp formation, particularly with ST1 and wild ST3. Among the polyp inducers, high numbers of mast cells were detected in wild ST3 and ST4, while a low number was found with heterozygous ST3. The level of secretory IgA was low in polyp-inducing STs. Most of the results were statistically significant. CONCLUSION: Immunosurveillance showed a potential relationship between ST1 and the ST3 intrasubtypes and precancerous polyps. This relationship may provide insight into the prevention and/or development of new immunotherapeutic strategies to combat colorectal cancer.

Evaluation of the effect of some medicinal plants on cultured Trichomonas Vaginalis.

INTRODUCTION: Trichomoniasis is a worldwide sexually transmitted disease caused by Trichomonas vaginalis. It inflicts severe complications to the human genitourinary system. The devastating negative effects and the emergence of resistance to common medication impose the search for safer and effective alternatives. This research aimed to investigate the effect of the Allium sativum, Nigella sativa crude extracts (NsCE) and the combination between their most effective doses with metronidazole. METHODOLOGY: Vaginal swabs were obtained from symptomatic patients, and cultured on Diamond's medium. Assessment of various concentrations of these herbs at different follow-up periods was done by counting the number of dead T. vaginalis trophozoites using the hemocytometer and trypan blue staining. Transmission electron microscope study was done. RESULTS: NsCE 9 mg/mL yielded the highest lethal effect on T. vaginalis trophozoites after 72 hours, compared with metronidazole. Combination of NsCE 9 mg/mL and metronidazole 50 µg/mL gave the best result. Additionally, Tomex90 µg/mL, represents a tolerable effect after 72 hours, but metronidazole 100 µg/mL still has higher effect. These results were confirmed by the ultrastructural changes observed in T. vaginalis trophozoites, signifying severe damage of nucleus and cytoplasm with large vacuolization and cell membrane defects. CONCLUSIONS: NsCE is a promising anti-Trichomonas especially its combination with metronidazole which showed a high synergistic effect.

Deciphering pyrethroid resistance in Cx. pipiens (L): Implications of cytochrome P450; expression profiling and regulatory microRNA.

Over the past decades, the extensive use of pyrethroids insecticides for vector control has resulted in the development of insecticide resistance. Cytochrome P450 has been recognized to play a critical role in the metabolic detoxification of insecticides. In the current study, Culex pipiens mosquitoes were collected from Giza Governorate in Egypt and tested for insecticide susceptibility against deltamethrin. First detection of Knockdown resistance gene (Kdr) mutations in field collected mosquitoes was performed. Activities of cytochrome oxidase P450 detoxification enzyme that synchronized with the resistance development, was assessed. Expression profiles of cytochrome P450s and their putative corresponding regulating miRNAs, which was previously reported in Cx. pipiens pallens were evaluated in pyrethroid resistant field-collected Cx. pipiens using RT-qPCR and stem-loop RT-qPCR, respectively. Specific stem-loop reverse transcription primers and forward primers were designed for miRNAs profiling. Our results elucidated the pyrethroid resistance development and revealed its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection. RT-qPCR results have showed an up-regulation in the expression of the studied P450 transcripts. Negative correlations were found between the expression of P450s and their regulatory miRNAs except for CYP9J35, where positive correlation was found with its corresponding miR-13. Interestingly, our data was the first to detect negative correlation between miR-285 and its putative CYP6Cp1 target gene. These findings highlighted the significance of identifying P450 gene along with regulatory miRNAs as a key mechanism implicated in pyrethroid resistance in field Culex vector population. The elucidation of this mechanism would shed light on the development of insecticide resistance and would help in shaping strategies to combat such vectors.

Performance of 2 Polymerization Protocols Targeting Cloned Toxoplasma Parasites.

BACKGROUND: Toxoplasma gondii is a common parasitic infection of humans. Infection is usually mild. Serious complications can occur in pregnant and immunocompromised patients. AIM: The present study aims to investigate the performance of 2 different PCR protocols; real-time quantitative molecular assays (qPCR) and conventional molecular assays (cPCR), using 2 different sets of primers and by using cloned purified Toxoplasma genomic substances to be evaluated as reference samples. METHODS: The target DNA was provided in 8 different quantities. RESULTS: Amplification failure was reported only with the cPCR in samples of low concentrations using both primer sets. Quantitative PCR detected the 8 different dilutions of the purified Toxoplasma gondii using the 2 sets of primers while cPCR was sensitive to detect only 6 different dilutions. CONCLUSION: Generally real-time quantitative molecular assays, is easy to use method compared to conventional PCR assay and produces more reliable results within only one hour time but still the possible application of qPCRs in routine diagnosis necessitates analysis of a large number of clinical samples in further studies to make the proper choice.

Isothermal PCR for Feasible Molecular Diagnosis of Primary Toxoplasmosis in Women Recently Experienced Spontaneous Abortion.

AIM: The current study aimed to assess the practicability of a simple loop-mediated isothermal amplification (LAMP) about real-time quantitative PCR to diagnose primary toxoplasmosis among high-risk pregnant women. METHODS: Cloned Toxoplasma samples were used to calculate the analytical sensitivity while specificity was assessed using pooled DNA samples extracted from other parasitic stages. RESULTS: Both techniques showed 100% sensitivity and specificity and then applied to detect recent Toxoplasma infection in peripheral blood of 77 IgG negative women out of a total 139 women lately experienced spontaneous abortion. The 2 techniques obtained positive results in 8 samples confirming primary toxoplasmosis. CONCLUSION: Generally, LAMP assay is a simple, cost-effective molecular technique can be completed in less than half an hour to diagnose primary Toxoplasma infection. The technique can be applied in a minimally equipped laboratory by ordinary workers to screen the vulnerable groups. Further analysis using larger samples with the quantitative approach is recommended to confirm the sensitivity of this emergent molecular technique.

GENETIC POLYMORPHISM OF TOXOPLASMA GONDII FROM RECENTLY INFECTED ABORTED EGYPTIAN WOMEN.

Toxoplasmosis caused by Toxoplasma gondii is one of the most prevalent parasitic diseases in human beings. Human toxoplasmosis can be associated with serious clinical manifestations, particularly in developing fetus. The aim of the current study was to identify the possible lineage type of Toxoplasma gondii, molecularly detected in placental samples of women whose pregnancies were spontaneously terminated in the first trimester. Preliminary detection of Toxoplasma genomic materials was done by a SYBR green qPCR technology. Subsequent identification of Toxoplasma strain was done for the positive samples using PCR-restriction fragment length polymorphism (RFLP) at the SAG2 loci of T. gondii using restriction enzymes HhaI and Sau3AI. Out of 72 tested samples, Toxoplasma B1 gene was detected in 9 cases. Toxoplasma genotypes I and II in addition to unknown type were identified in 4, 3 and 2 cases respectively, while type III was not detected in our samples, hence excluded as a leading cause of abortion in humans in our preliminary study. Nevertheless, it remains uncertain to what extent the genotype of the parasite directly contributes to the clinical severity of human toxoplasmosis. Certainly, advanced molecular techniques targeting different Toxoplasma strains are crucial for better understanding of human toxoplasmosis. For more elucidation, additional studies are recommended intended for genetic characterization of such serious parasitic infection using larger number of samples.

LIMIT OF MOLECULAR DETECTION OF STRONGYLOIDES STERCORALIS PARASITE PERFORMING.TWO PCR PROTOCOLS.

Low parasite density in chronic infection with S. stercoralis is a challenging diagnostic issue. Generally, molecular techniques don't depend on parasite viability while copro-culture or Baermann concentration method relies on the presence of living larvae in fecal samples. Therefore, evaluation of PCR-based methods is important to increase the detection rates in light or chronic infection. This study was designed to analyze the sensitivity of quantitative PCR (qPCR) and nested PCR (nPCR) regarding the minimum amount of S. stercoralis DNA template that can be reliably detected by each molecular technique. Strongyloides larvae were collected from cultured stool samples from suspected infected Egyptian children. After counting larvae present in a known volume under the microscope, DNA extraction was done and se- rial dilution of genomic materials was prepared. Then, qPCR and nPCR targeting the, small subunit of the rRNA gene were performed. Regarding qPCR, the limit of detection was 0.0005 S. stercoralis larvae/µl, with crossing threshold (Ct) Value ranged from 17.8 to 38.7 while, nPCR did not detect from (0.002 to 0.0012 S tercopilis larvae/µl) with minimum limit 0.004 S. stercoralis larvae/µl. Real-time quantitative PCR is very sensitive technique that can detect very low genomic load up to about 10 (9.765) genome copies/reaction compared to. nested PCR which started positivity from 78.125 genome copies/reaction. Therefore, qPCR is recommended to detect chronic strongyloidiasis especially in high-risk groups to prevent life- threatening spread of such infection.