Comparing cytotoxicity and efficacy of miltefosine and standard antimicrobial agents against Acanthamoeba trophozoites and cyst forms: An in vitro study.

Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse effects. Our study aimed to compare the in vitro activities and safety of Miltefosine with that of conventional antimicrobial agents used to treat AK. Acanthamoeba castellanii genotype T4 was obtained from a patient with keratitis and subjected to in vitro susceptibility testing with various antimicrobial agents, including Chlorhexidine (CHX), Pentamidine isethionate (PI)Polyhexamethylene biguanide (PHMB), and Miltefosine to assess their efficacy against Acanthamoeba trophozoites and cyst. The cytotoxicity of the agents was evaluated in Vero cells, and their selectivity indexes (SI) were calculated. Chlorhexidine exhibited the highest amoebicidal activity with the highest selectivity index against the trophozoite and cyst, ranging from 1.17 to 8.35. The selectivity index of PHMB is slightly comparable to Chlorhexidine, exhibiting significant anti-Acanthamoeba activity. On the other hand, Pentamidine isethionate and Miltefosine displayed low SI among the compounds. Pentamidine isethionate was effective at high concentrations, which was toxic. Miltefosine exhibited the lowest cytotoxicity; nevertheless, due to the lowest anti-Acanthamoeba activity presented a low selectivity against the parasite. Further studies on more clinical samples and prolonged incubation time should be done to investigate the effectiveness and toxicity of drugs in both in vitro and in vivo conditions.

In Vitro Activity of Pentamidine Isethionate against Trophozoite and Cyst of Acanthamoeba.

BACKGROUND: Acanthamoebae are a causative agent of Acanthamoeba keratitis (AK) in immunocompetent individuals. Since access to propamidine isethionate (Brolene®) as a first-line treatment has been limited in recent years, in the current study, we examined the effects of pentamidine isethionate against trophozoite and cyst forms of Acanthamoeba. METHODS: This experimental study was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, during 2019-2020. Pentamidine isethionate at concentrations of 50, 100, 200, 400, 600, 800, and 1000 µM were tested against trophozoites and cyst stages of T4 genotype, at 24- and 48-hour incubation period, and the viability was determined by trypan blue staining. In addition, the cytotoxic effect of the drug was examined in Vero cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The 50% inhibitory concentration (IC50) of pentamidine isethionate on trophozoite after 24 and 48h were 97.4 µM and 60.99 µM. These results on cyst after 24 and 48h were 470 µM and 175.5 µM, respectively. In MTT assay, the drug showed an inhibitory effect on Vero cell growth with IC50 values of 115.4 µM and 87.42 µM after 24h and 48h, respectively. CONCLUSION: Pentamidine isethionate exhibited an inhibitory effect on trophozoite and cyst. Given that the trophozoicidal activity of the drug is in the safe dose, it could be suggested as an alternative in patients with AK; however, further investigation is needed in an animal model to confirm the data.

Sarcocystis infection in beef and industrial raw beef burgers from butcheries and retail stores: A molecular microscopic study.

Sarcocystis is a genus of eucoccidian parasites, which globally infects humans and various animals. In addition to economic losses in livestock industries, the parasite is a zoonosis that infects humans through contaminated beef and pork with the parasite sarcocysts. Therefore, this study was carried out to assess Sarcocystis contamination in beef and industrial raw beef burger samples from butcheries and retail stores in Tehran, Iran. Overall, 180 samples of 90 beefs and 90 raw industrial beef burgers with at least 80% meat were randomly collected in Tehran, Iran. Samples were studied microscopically after peptic digestion. Furthermore, sample genomic DNAs were used in conventional polymerase chain reaction (PCR) to amplify approximately 900-bp fragments from 18S ribosomal DNA. Of 180 samples, 170 samples (94.4%) were microscopically and 161 samples (89.44%) were molecularly positive for Sarcocystis spp. Eucoccidial DNA fragments were detected in 161 samples (89.4%), including 78 (86.6%) beef and 83 (92.2%) beef burger samples. No significant differences were found between the beef and beef burger infestations by Sarcocystis bradyzoites using statistical analysis (P > 0.05). Statistically significant differences were seen between the sample type and the intensity of parasites in samples (P = 0.003). Furthermore, differences between the conventional PCR results (positive/negative) and the intensity of parasites in samples were statistically significant (P < 0.001). The considerable prevalence of Sarcocystis spp. in beef and beef burger samples reflects high transmission of the parasite in meat producing cattle, which is important due to food hygiene. Although the most prevalent bovine species, S. cruzi, is not a zoonosis, it is highly recommended to follow guidelines on the parasite transmission prevention due to the existence of S. hominis as a zoonotic bovine species.

Development, optimization, and validation of an in-house Dot-ELISA rapid test based on SAG1 and GRA7 proteins for serological detection of Toxoplasma gondii infections.

BACKGROUND: The aim of the present study was to develop a simple, portable, and rapid assay for serodiagnosis of toxoplasmosis based on recombinant Toxoplasma gondii (T. gondii) SAG1 (rSAG1) and GRA7 (rGRA7) proteins. METHODS: The rSAG1 and rGRA7 proteins were expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. The immunoreactivity of the recombinant antigens was tested in an in-house IgG and IgM Dot enzyme-linked immunosorbent assay (Dot-ELISA) for potential use in serodiagnosis of T. gondii infection. RESULTS: Results from the comparison of in-house rSAG1-Dot-ELISA with ELISA for the detection of anti-Toxoplasma IgG and IgM include sensitivity of 83.7% and 81.2%, specificity of 90.2% and 89.3%, positive predictive values of 85.9% and 68.4%, and negative predictive values of 88.6% and 94.3%, respectively. Sensitivity of 66.2%, specificity of 81.2%, positive predictive values of 71.6%, and negative predictive values of 77.1% were concluded from in-house IgG rGRA7-Dot-ELISA. The sensitivity and specificity of IgM rGRA7-Dot-ELISA included 87.5% and 83.9%, respectively. Sensitivity and specificity of in-house Dot-ELISA for a combination of rSAG1 and rGRA7 included 87.5% and 91.1% for IgG and IgM, respectively. Sensitivity and specificity of a combination of rSAG1 and rGRA7 for the detection of IgM in suspected sera to acute toxoplasmosis were higher than those for the detection of IgG in sera with chronic infections (90.6% and 92% instead of 86.2% and 91.6%, respectively). CONCLUSION: The highlighted parameters of combined recombinant proteins were more significant than those of single recombinant proteins in in-house Dot-ELISA. These data suggest that the in-house Dot-ELISA based on rSAG1 and rGRA7 combination is a promising diagnostic tool with a similar sensitivity to the native antigens of T. gondii, which can be used for the serodiagnosis of toxoplasmosis in fields as well as less equipped laboratories.

Detection of toxoplasma-specific immunoglobulin G in human sera: performance comparison of in house Dot-ELISA with ECLIA and ELISA.

In the current study, performance of electrochemiluminescence immunoassay (ECLIA) in detection of anti-toxoplasma IgG in human sera was compared with that of enzyme-linked immunosorbent assay (ELISA). Furthermore, performance of an in house Dot-ELISA in detection of anti-toxoplasma IgG was compared with that of ECLIA and ELISA. In total, 219 human sera were tested to detect anti-toxoplasma IgG using Dynex DS2® and Roche Cobas® e411 Automated Analyzers. Discordant results rechecked using immunofluorescence assay (IFA). Then, sera were used in an in house Dot-ELISA to assess toxoplasma-specific IgG. Of the 219 samples, two samples were found undetermined using ECLIA but reactive using ELISA. Using IFA, the two sera were reported unreactive. Furthermore, two samples were found reactive using ECLIA and unreactive using ELISA. These samples were reported reactive using IFA. The overall agreement for the two former methods was 98% (rZ0.98.1; P < 0.001). The intrinsic parameters calculated for in house Dot-ELISA included sensitivity of 79.5, specificity of 78.2, and accuracy of 78.9%, compared to ECLIA and ELISA. Positive and negative predictive values included 82.9 and 74.2%, respectively. A 100% sensitivity was found in in house Dot-ELISA for highly reactive sera in ECLIA and ELISA. ECLIA is appropriate for the first-line serological screening tests and can replace ELISA due to high speed, sensitivity, and specificity, particularly in large laboratories. Dot-ELISA is a rapid, sensitive, specific, cost-effective, user-friendly, and field-portable technique and hence can be used for screening toxoplasmosis, especially in rural fields or less equipped laboratories.

Severe diarrhea due to Isospora belli in a patient with thymoma.

Opportunistic isosporidial infection of the gastrointestinal tract is frequently encountered in patients with acquired immunodeficiency syndrome (AIDS) and is considered to be an AIDS-defining illness. Chronic severe watery diarrhea due to Isospora belli has also been reported in other immunodeficiency states. This report describes severe chronic debilitating diarrhea due to isosporiasis in a patient with mediastinal thymoma, a common tumor of the anterior mediastinum, originating from the epithelial cells of the thymus. Numerous oocysts of I. belli were detected in direct smear preparation of the diarrheic stool sample of the patient, who had an 8-month history of recurrent diarrhea. Duodenal and colonic mucosal biopsies revealed slight degrees of atrophic changes associated with infiltration of the lamina propria by an appreciable number of eosinophiles and the presence of unizoit tissue cysts of I. belli in the surface epithelium of the duodenal mucosa. The patient was first treated with trimethoprim-sulfamethoxazole and subsequently underwent complete thymectomy. Later, due to recurrence of the diarrhea, he was treated with ciprofloxacin.

Molecular characterization of Cryptosporidium isolates from humans and animals in Iran.

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.

Genetic diversity of glucose phosphate isomerase from Entamoeba histolytica.

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucose phosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, IIalpha-, XIV, or XIX. Two alleles were obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eight polymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points were identified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolytica and other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterization of recombinant E. histolytica GPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility of three GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patterns that have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.

Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran.

The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities.

Cryptosporidium parvum bovine genotype oocysts in the respiratory samples of an AIDS patient: efficacy of treatment with a combination of azithromycin and paromomycin.

Cryptosporidium has been recognized as an emerging zoonotic agent of intestinal cryptosporidiosis leading to diarrhea, malabsorption syndrome, and weight loss in AIDS patients. In the present case, oocysts of zoonotic Cryptosporidium parvum were detected in the sputum and stool samples of an AIDS patient with a 3-month history of intestinal cryptosporidiosis. The oocysts were detected by modified Ziehl-Neelsen staining; confirmation was achieved by nested polymerase chain reaction (PCR), targeting the most polymorphic region of the 18S rRNA gene. Genotyping was done by restriction endonuclease digestion of the PCR product. The zoonotic C. parvum bovine genotype was identified in both intestinal and respiratory samples. Treatment with both azithromycin and paromomycin resulted in improvement of both intestinal and respiratory symptoms, as well as the elimination of the parasite. This is the first report of the identification of Cryptosporidium sp. oocysts in the respiratory samples obtained from an AIDS patient in Iran. Pulmonary cryptosporidiosis should be considered whenever an AIDS patient with intestinal cryptosporidiosis develops respiratory symptoms.

Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates.

The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5%) belonged to T4, two (15.3%) belonged to T3 and three (23%) belonged to the T2 genotype. In contrast, the majority of 12 environmental isolates tested in the present study belonged to T2 (7/12, 58.3%), followed by 4/12 T4 isolates (33.3%). In addition, the genotypes of six new Acanthamoeba isolates from UK keratitis cases were determined. Of these, five (83.3%) belonged to T4 and one was T3 (16.6%), supporting the expected high frequency of T4 in Acanthamoeba keratitis. In total, the genotypes of 24 Acanthamoeba keratitis isolates from the UK and Iran were determined. Of these, 17 belonged to T4 (70.8%), three belonged to T2 (12.5%), three belonged to T3 (12.5%) and one belonged to T11 (4.1%), confirming that T4 is the predominant genotype (S2=4.167; P=0.0412) in Acanthamoeba keratitis.

Prevalence of intestinal parasitic pathogens among HIV-positive individuals in Iran.

Parasites are important enteric pathogens among patients with human immunodeficiency virus (HIV) infection. There have been very few reports on the prevalence of intestinal parasites among such patients in Iran. To determine the prevalence of intestinal parasites among HIV-positive individuals, we collected single stool samples and analyzed them for detection of various intestinal parasites from 206 HIV-positive individuals with different immune status visited in different medical centers in Iran. The data were tested for statistical significance with chi(2) and Mann-Whitney U tests. The overall prevalence of intestinal parasites was 18.4% (95%CI: 13.7, 24.3). More specifically, the following parasites were identified: Giardia lamblia (7.3%), Blastocystis hominis (4.4%), Entamoeba coli (3.9%), and Cryptosporidium parvum (1.5%). Other parasites observed included Strongyloides stercoralis and Hymenolepis nana in two cases and Dicrocoelium dendriticum in one. Of the 38 patients who tested positive for intestinal parasites, 15 (39.2%) had diarrhea. Intestinal parasites were significantly more common among patients with diarrhea than those without (P < 0.001). Further, CD4 counts were significantly lower among individuals with diarrhea than those without (P < 0.001). This study highlights the importance of testing for intestinal parasites among Iranian HIV-positive patients, especially those with low immunity presenting with diarrhea.

Distribution and differential diagnosis of Entamoeba histolytica from Entamoeba dispar by the PCR-RFLP method in Central Iran.

BACKGROUND: Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable human protozoan parasites that are genetically distinct species. The potential invasive pathogenic Entamoeba histolytica and non-invasive parasite Entamoeba dispar can be differentiated by molecular and other methods. We used polymerase chain reaction (PCR) to determine the ratio of the two species in a population in Tehran and Karaj in central Iran. MATERIALS AND METHODS: Human stool samples (n=12 148) were randomly collected in Tehran and Karaj and examined for E. histolytica/E. dispar cysts with direct and formalin-ether methods. Eighty-seven (0.7%) cases were positive, of which 49 (62.8%) isolates were successfully cultured in Robinson s medium. A pair of oligonucleotide primers designed from sequence data for genomic DNA coding the 30-KD surface antigen of E. histolytica/E. dispar was used to amplify a 374 base-pair (bp) fragment. The electrophoretic pattern of the PCR product digested with Hinfl restriction enzyme was used for differentiation of the two species. RESULTS: The restriction fragment length polymorphism (RFLP) pattern obtained from a standard E. histolytica isolate had two fragments (219 bp and 155 bp), but the standard isolate of E. dispar showed three fragments (155, 152 and 67 bp). Differential diagnosis of 49 isolates of E. histolytica/E. dispar from Tehran and Karaj using PCR-RFLP revealed that 46 (93.9%) were E. dispar while only 2 (4.1%) were E. histolytica. One person (2%) had a mixed infection and showed both patterns. CONCLUSION: The differential diagnosis of the potentially pathogenic parasite E. histolytica from the non-pathogenic E. dispar is of clinical and epidemiological importance. This study demonstrated that E. dispar is much more prevalent than E. histolytica among the cyst passers in Tehran and Karaj in Central Iran.