Nanoformulation-Based 1,2,3-Triazole Sulfonamides for Anti-Toxoplasma In Vitro Study.

Toxoplasma gondii is deemed a successful parasite worldwide with a wide range of hosts. Currently, a combination of pyrimethamine and sulfadiazine serves as the first-line treatment; however, these drugs have serious adverse effects. Therefore, it is imperative to focus on new therapies that produce the desired effect with the lowest possible dose. The designation and synthesis of sulfonamide-1,2,3-triazole hybrids (3a-c) were performed to create hybrid frameworks. The newly synthesized compounds were loaded on chitosan nanoparticles (CNPs) to form nanoformulations (3a.CNP, 3b.CNP, 3c.CNP) for further in vitro investigation as an anti-Toxoplasma treatment. The current study demonstrated that all examined compounds were active against T. gondii in vitro relative to the control drug, sulfadiazine. 3c.CNP showed the best impact against T. gondii with the lowest IC50 value of 3.64 µg/mL. Using light microscopy, it was found that Vero cells treated with the three nanoformulae showed remarkable morphological improvement, and tachyzoites were rarely seen in the treated cells. Moreover, scanning and transmission electron microscopic studies confirmed the efficacy of the prepared nanoformulae on the parasites. All of them caused parasite ultrastructural damage and altered morphology, suggesting a cytopathic effect and hence confirming their promising anti-Toxoplasma activity.

Sulfadiazine analogs: anti-Toxoplasma in vitro study of sulfonamide triazoles.

Toxoplasmosis is an infection that prevails all over the world and is caused by the obligate intracellular protozoan parasite Toxoplasma gondii (T. gondii). Promising novel compounds for the treatment of T. gondii are introduced in the current investigation. In order to test their in vitro potency against T. gondii tachyzoites, six 1,2,3-triazoles-based sulfonamide scaffolds with terminal NH2 or OH group were prepared and investigated as sulfadiazine equivalents. When compared to sulfadiazine, which served as a positive control, hybrid molecules showed much more anti-Toxoplasma activity. The results showed that the IC50 of the examined compounds 3(a-f) were recoded as 0.07492 µM, 0.07455 µM, 0.0392 µM, 0.03124 µM, 0.0533 µM, and 0.01835 µM, respectively, while the sulfadiazine exhibited 0.1852 µM. The studied 1,2,3-triazole-sulfadrug molecular conjugates 3(a-f) revealed selectivity index of 10.4, 8.9, 25.4, 21, 8.3, and 29; respectively. The current study focused on the newly synthesized amino derivatives 3(d-f), as they contain the more potent amino groups which are recognized to be essential elements and promote better biological activity. Extracellular tachyzoites underwent striking morphological alterations after 2 h of treatment as seen by scanning electron microscopy (SEM). Additionally, the intracellular tachyzoite exposed to the newly synthesized amino derivatives 3(d-f) for a 24-h period of treatment revealed damaged and altered morphology by transmission electron microscopic (TEM) indicating cytopathic effects. Moreover, compound 3f underwent the most pronounced changes, indicating that it had the strongest activity against T. gondii.

Molecular diagnosis and subtyping of Blastocystis sp.: Association with clinical, colonoscopic, and histopathological findings.

Introduction: Blastocystis sp. is the most common parasitic infestation in humans. However, its pathogenicity remains controversial. Our aim was to study the prevalence of Blastocystis sp. parasite subtypes in patients with gastrointestinal manifestations referred for colonoscopy and assess possible correlation with clinical, colonoscopic, and histopathological findings. Methodology: One hundred patients with gastrointestinal manifestations referred for colonoscopy were enrolled. Stool samples were collected and examined both microscopically and by real-time quantitative polymerase chain reaction (qPCR) for detection of Blastocystis sp. Subtyping was done for positive samples by qPCR and confirmed by sequencing. Results: qPCR sensitivity far exceeded microscopy in detection of Blastocystis sp. (58% vs. 31%, agreement 38.5%). The most commonly detected subtype was 3 (50%), followed by 2 (32.8%) and 4 (13.8%). Abdominal pain was the most common clinical symptom; inflammation and colitis were the most common abnormal colonoscopic and histopathological findings. The most frequent subtype encountered in those findings was Subtype 3. Conclusions: This study confirmed the importance of using qPCR in diagnosis of Blastocystis sp. An association between abnormal clinical, colonoscopic, and histopathological findings on the one hand, and Blastocystis sp. infestation, especially Subtype 3, on the other hand, is also posed. This necessitates further studies to assess the mechanism of association with pathogenicity.

Proposed morbidity markers among Schistosoma mansoni patients.

Background: Fecal calprotectin (FC) and fecal occult blood (FOB) were suggested as potential inflammatory markers for assessing intestinal schistosomiasis morbidity that are conventionally detected through invasive methods. Aim and Objectives: The present work aimed to evaluate FC and FOB as morbidity markers of Schistosoma mansoni infection before and after praziquantel treatment. Materials and Methods: A total of 205 stool samples (117 schoolchildren and 88 adults) were collected and examined by Kato Katz. A questionnaire enquiring about diarrhea, history of blood in stool, and abdominal pain was designed and applied. Results: S. mansoni prevalence rates were 20.5% and 11.36% among children and adults, respectively; the majority of cases had light infection intensity. FC and FOB were studied among 25 cured S. mansoni cases (17 children and 8 adults) pre and one-month post treatment. Before treatment, six and four children of moderate and high S. mansoni infection intensity tested positive for FC and FOB, respectively, all turning negative after treatment. FC showed borderline statistical significance before and after treatment among children. However, all adults tested negative for FC and FOB. Conclusion: FC and FOB could be possibly used as morbidity monitoring tools for S. mansoni infection in children with moderate and high infection intensity.

Epidemiological profile of microbial keratitis in Alexandria-Egypt a 5 years retrospective study.

OBJECTIVE: To evaluate the epidemiologic profile of microbial keratitis in Alexandria- Egypt, with special emphasis on risk factors, visual outcome and microbiological results. METHODS: This retrospective study reviewed files of patients treated for microbial keratitis during a period of 5 years at Alexandria Ophthalmology Hospital Cornea Clinic, Alexandria- Egypt, between February 2017 and June 2022. The patients were evaluated for the risk factors e.g., trauma, eyelid disorders, co-morbidities, and contact lens use. They were also evaluated for their clinical picture, the identified microorganisms, visual outcomes, and complications. Non-microbial keratitis and incomplete files were excluded from the study. RESULTS: A total of 284 patients were diagnosed as microbial keratitis in our study. Viral keratitis was the most common cause of microbial keratitis (n = 118 (41.55%)), followed by bacterial keratitis (n = 77 (27.11%)), mixed keratitis (n = 51 (17.96%)), acanthamoeba keratitis (n = 22 (7.75%)) and the least cause was fungal keratitis (n = 16 (5.63%)). Trauma was the most common risk factor for microbial keratitis (29.2%). Fungal keratitis had a statistically significant association with trauma (p < 0.001), while the use of contact lenses had a statistically significant association with Acanthamoeba keratitis (p < 0.001). The percentage of culture-positive results in our study was 76.8%. Gram-positive bacteria were the most frequently isolated bacterial isolate (n = 25 (36.2%)), while filamentous fungi were the most frequently isolated fungi (n = 13(18.8%)). After treatment, there was a significant increase in the mean visual acuity among all groups; it was significantly higher in Acanthamoeba keratitis group with a mean difference of 0.262 ± 0.161 (p = 0.003). CONCLUSION: Viral keratitis followed by bacterial keratitis were the most frequent etiologic agents causing microbial keratitis found in our study. Although trauma was the most frequent risk factor for microbial keratitis, contact lens wear was found an important preventable risk factor for microbial keratitis in young patients. Performing culture properly whenever indicated before starting antimicrobial treatment increased the cultures' positive results.

Evaluation of microscopy and PCR for detection of Dientamoeba fragilis.

Introduction: Dientamoeba fragilis (D. fragilis) diagnosis is an intestinal protozoan parasite globally found in rural and urban areas and is attracting a growing interest. Its prevalence in stool varies from 0.2% to more than 19% depending upon the population studied. Materials and Methods: This study was based on the examination of 100 stool samples of randomly referred cases in a rural area in Motobus district, Kafr El-Sheikh governorate, Egypt. Our aim was to investigate the presence of D. fragilis in stool of the examined individuals using conventional polymerase chain reaction (PCR) compared to wet mount and trichrome stain with confirmation of infection by transmission electron microscopy. Results: D. fragilis was detected in 13/100 of the stool samples examined using wet mount smears, while trichrome stain detected 17/100. Conventional PCR diagnosed 41 cases of D. fragilis in the studied group. A very good agreement was found between wet mount and trichrome stain for diagnosing D. fragilis, while there was fair agreement between conventional PCR and both microscopy methods. Transmission electron microscope was performed on pooled positive samples that revealed the internal structures of D. fragilis trophozoite with its characteristic nucleus. Conclusions: PCR technique was superior to microscopy for the detection of D. fragilis. Trichrome stain remains vital for microscopic diagnosis.

Detection and Molecular Identification of Cryptosporidium Species Among Children with Malignancies.

BACKGROUND: Cryptosporidiosis represents a major health problem worldwide particularly among children. Its diagnosis is still difficult and demands sensitive methods. In Egypt, there is little documentation of infection among children with malignancies. This work was designed to study the infection rate of Cryptosporidium among children with malignancies, compare the performance of modified Ziehl-Neelsen (MZN) stain with nested polymerase chain reaction (PCR) and identify the species subtypes of positive cases. METHODS: The study was conducted on 100 children with malignancies (leukemia, lymphoma and solid tumors), below 10 years of age, from El-Shatby hospital, Alexandria University. After obtaining the informed consent, their stool samples were collected and examined microscopically following MZN stain for the diagnosis of Cryptosporidium spp. All samples were then subjected to nested PCR. Restriction fragment length polymorphism (RFLP) targeting the Cryptosporidium oocyst wall protein (COWP) gene was applied to positive cases, using restriction enzyme RsaI for digestion of nested PCR products. RESULTS: Out of the 100 examined children, MZN detected higher positive cases compared to nested PCR. Six cases (6%) were diagnosed positive by MZN stain, three of which (3%) were concordantly positive by nested PCR. All positives were among children with acute lymphoblastic leukemia (ALL). Fair agreement was found between the two tests (K = 0.36). Genotyping results revealed that positive samples were of Cryptosporidium parvum (C. parvum) type II. CONCLUSIONS: Low Cryptosporidium infection rate was detected among children with malignancies. MZN diagnosed more positive cases compared to nested PCR. C. parvum type II was the identified species among the examined children. Further optimization of PCR steps is needed.

Intestinal schistosomiasis: Can a urine sample decide the infection?

Intestinal schistosomiasis, one of the neglected tropical diseases whose control depends on accurate diagnosis of the disease prevalence. The use of low sensitive Kato Katz (KK) fecal egg detection method as a reference gold standard is not an accurate indication especially in low transmission areas. Latent class analysis frameworks especially the Bayesian could be used instead to compare between different diagnostic tests without the use of a gold standard method as a reference. Thus, this study compared two urine-based tests for the detection of circulating antigen and cell free DNA of Schistosoma mansoni versus KK method using the Bayesian latent class analytical framework and in two models where the trace results of point of contact - assay of circulating cathodic antigen (POC-CCA) were once estimated as positive, and as negative in the other model. The Bayesian framework in the trace CCA positive model showed an estimate of disease prevalence of 26% (95% BCI:0 to 60%). POC-CCA showed the highest sensitivity (74% with BCI: 9 to 91%) and lowest specificity for (20% with BCI: 0% to 37%) and the reverse for KK. For POC-CCA with traces considered negative, it was found that results between the three tests were moderated where the positivity for infection by Schistosoma antigen detection and PCR for cell free DNA approached that estimated by the Bayesian framework (44%), and the specificity for point of contact assay(81%; 95%BCI: 59% to 100%) rose in hand with its sensitivity(77%, 95% BCI:53% to 100%) and with results for PCR test (sensitivity = 80%; 95% BCI: 61% to 100%, specificity = 69%; 95% BIC: 47% to 100%). KK remains with the highest specificity while its sensitivity in the two models never exceeded 22%. Thus, we conclude that the use of a single urine sample could be very sensitive and highly specific in the diagnosis of intestinal schistosomiasis using either the trace negative model of point of contact assay, or conventional PCR, when compared to the fecal egg detection using duplicate KK. However, the use of a single tool restricts the management of the disease in areas of low endemicity.

Intestinal Microsporidia Infection in Leukemic Children: Microscopic and Molecular Detection.

BACKGROUND: Microsporidia infection was originally described as an immunocompromised associated pathogen. Limitations to correct microscopic diagnosis of microsporidia include size of the organism presenting a challenge even to a highly competent laboratory expert. OBJECTIVE: The present study aimed to detect microsporidia infection among leukemic children. The performance of modified trichrome stain and PCR in the diagnosis of microsporidia was evaluated with further speciation. METHODS: Stool samples of 100 leukemic children on chemotherapy were examined microscopically for microsporidia. DNA was extracted from all samples. Amplification was performed by conventional and nested PCR. Sequencing of amplified products was performed on unspeciated samples. RESULTS: Microsporidia were detected in 23% of the children by MTS and 29% by PCR. The 29 positive samples were subjected to PCR for speciation. Enterocytozoon bieneusi was found to predominate in 20 cases, Encephalitozoon intestinalis in three cases, two cases had co-infection, and the remaining four samples were not amplified with either E. bieneusi or E. intestinalis specific primers. By DNA sequencing of the unspeciated samples, three samples shared high homology with Encephalitozoon hellem and one sample with Encephalitozoon cuniculi. Referring to PCR as a gold standard, MTS exhibited 72.4% sensitivity and 97.2% specificity with 90% accuracy. Among a number of studied variables, diarrhea and colic were significantly associated with microsporidia infection when diagnosed by either technique. CONCLUSION: The use of sensitive and discriminative molecular tools will contribute to determining the true prevalence of microsporidiosis and possibly their potential transmission source depending on species identification.

Eristalis tenax intestinal myiasis: An electron microscope study.

Myiasis is the infestation of live vertebrates (humans or animals) with dipterous larvae. Eristalis tenax, belonging to order Diptera and family Syrphidae, seldom causes accidental myiasis, usually due to ingestion of contaminated food or water by humans. Here, we report a case of intestinal myiasis in a male from Alexandria, Egypt, complaining of frequent passage of small worms in his stool. A larva and a pupa were presented to the laboratory and examined macroscopically, and then studied by a scanning electron microscope. E. tenax (rat-tailed maggots) were diagnosed. Rarely diagnosed worldwide, a case of E. tenax accidental intestinal myiasis was found in a middle-aged adult male from Egypt. A larva and a pupa were identified and studied macroscopically and by scanning electron microscope.

Improving Hashimoto's thyroiditis by eradicating Blastocystis hominis: Relation to IL-17.

BACKGROUND: Hashimoto's thyroiditis (HT) is a common autoimmune disorder that causes significant morbidity. Interleukin (IL)-17 was identified as a major contributing factor in the pathogenesis of HT. Blastocystis hominis (BH) is a very common infection and has been shown to be associated with several diseases. Our aim was to determine serum IL-17 level in HT patients with and without BH infection and the effect of eradicating BH in patients with HT. METHODS: A prospective cohort study was conducted on 20 HT patients not infected with BH (group I), 20 HT patients infected with BH (group II), and 20 healthy patients (group III). Serum free triiodothyronine (FT3), free thyroxine (FT4), thyroid stimulating hormone (TSH), thyroid peroxidase antibodies (anti-TPO), and IL-17 were performed by ELISA method and were repeated in group II after 6 weeks of eradication of BH. RESULTS: Patients with HT showed a significantly higher serum IL-17 compared with controls. IL-17 was significantly higher in HT patients infected with BH compared with HT patients not BH infected (mean 6.93 ± 2.83 pg/ml versus 3.25 ± 1.55 pg/ml, p = 0.003). After BH eradication TSH, anti-TPO, and IL-17 were significantly decreased (mean 14.76 ± 11.11 µIU/ml versus 9.39 ± 7.11 µIU/ml, p < 0.001; mean 308 ± 175.6 IU/ml versus 295.4 ± 167.1 IU/ml, p = 0.006; and mean 6.93 ± 2.83 pg/ml versus 6.45 ± 2.48 pg/ml, p < 0.001), respectively. Multivariate analysis after treating BH infection showed that IL-17 was significantly negatively correlated with FT3 (adjusted p = 0.002) and significantly positively correlated with anti-TPO (adjusted p = 0.045). CONCLUSION: Treatment of BH infection ameliorates HT through reduction in IL-17, anti-TPO, and TSH. CLINICAL TRIAL REGISTRATION NUMBER: PACTR201909495111649.

Successful treatment of acute experimental toxoplasmosis by spiramycin-loaded chitosan nanoparticles.

Spiramycin-metronidazole and spiramycin-loaded chitosan (CS) nanoparticles (NPs) were tested in comparison with the current spiramycin treatment of T.gondii concerning tissue penetration and blood brain barrier (BBB) passage. Swiss Albino mice were inoculated intraperitoneally with 2500 T. gondii tachyzoites RH strain and were divided into experimental and control groups. The experimental groups orally received CS NPs, spiramycin, spiramycin-metronidazole, spiramycin-loaded CS NPs 400 mg/kg and spiramycin-loaded CS NPs 100 mg/kg. Drug efficacy was assessed by mice survival time, mortality rate, parasite load in different organs and morphological study of the tachyzoites movement by light microscope and the ultra-structure by SEM. The results revealed that the maximum survival time of more than 200 days with no mortality on the sacrifice day (8th) was observed in mice receiving spiramycin-loaded NPs. Spiramycin-loaded NPs showed the highest significant percent reduction of tachyzoites (about 90% reduction) in liver, spleen and brain as compared to the other used drugs denoting successful bypass of BBB. Light microscopy of the treated peritoneal tachyzoites showed sluggish tachyzoites movement while the NPs caused loss of their movement. SEM of the treated tachyzoites were more mutilated and some of them appeared rupturing in those receiving CS NPs and spiramycin-loaded NPs. In conclusion, spiramycin-loaded NPs showed the highest efficiency in the treatment of acute toxoplasmosis. The non-toxic nature and the anti-parasitic effect of both CS and spiramycin make the use of spiramycin-loaded CS NPs a potential material for treatment of human toxoplasmosis.

Urinary circulating DNA and circulating antigen for diagnosis of schistosomiasis mansoni: a field study.

OBJECTIVE: To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village. METHODS: Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs. RESULTS: Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR. CONCLUSION: Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.

OBJECTIF: Evaluer trois tests non invasifs pour le diagnostic de la schistosomiase mansoni dans un village égyptien. MÉTHODES: L'urine a été collectée pour la détection de l'antigène cathodique circulant (ACC) et de l'ADN du parasite libéré des cellules (cfpd) par le test en cassette POC (point-of-contact) et par PCR, respectivement. Ces tests ont été comparés au test de Kato Katz (KK) sur frottis fécal épais pour la détection des œufs de Schistosoma mansoni. RÉSULTATS: La prévalence de la maladie par dosage POC-ACC était de 86%; elle était de 39% par PCR contre 27% par KK. Par rapport à KK, la sensibilité du POC-ACC atteignait 100%, mais sa spécificité n'était que de 19,2% avec une précision de 41%. La sensibilité du PCR pour la cfpd était de 55,56% et sa spécificité de 67,12% avec une précision de 64%. Un nouveau seuil a été calculé pour l'analyse combinée des tests KK, POC-ACC et PCR. La sensibilité pour les trois tests était de 52,94%, 90,2% et 76,47%; la spécificité était de 100% pour KK et PCR et de 18,37% pour POC-ACC. La précision calculée pour les trois tests au point seuil était de 76% pour KK, 55% pour le POC-ACC et 88% pour la PCR. CONCLUSION: Le test PCR conventionnel pour la détection de la cfpd constitue un outil de dépistage potentiel de la schistosomiase intestinale avec une spécificité fiable, une précision raisonnable et un coût financier et technique abordable.

Genotype analysis of T. gondii strains associated with human infection in Egypt.

The distribution of Toxoplasma gondii genotypes varies from one geographic area to another. The present study aimed to determine T. gondii genotypes associated with human infection in Egypt. Individuals seropositive for anti-toxoplasma IgG and IgM (group I, n = 50) or for specific IgG only (group II, n = 50) were enrolled. Of the participants, 75 % were asymptomatic pregnant women. The others presented with lymphadenitis (n = 21), chorioretinitis (n = 3), and unexplained hepatomegaly (n = 1). Using nested PCR, T. gondii GRA6-coding fragment was amplified from DNA extracted from blood samples of participants. Amplification was successful in 12 samples with nonsignificant difference between both groups but with a significant association with the presence of toxoplasmosis-related manifestations. Restriction fragment length polymorphism analysis of these samples revealed the presence of type I in seven samples and atypical types in five samples. Both typical and atypical strains were detected in individuals of both groups with no bias towards specific clinical presentation.