Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

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.

Detection of Enterobius vermicularis in archived formalin-fixed paraffin-embedded (FFPE) appendectomy blocks: It's potential to compare genetic variations based on mitochondrial DNA (cox1) gene.

Acute appendicitis represents one of the most common causes of emergency abdominal surgery worldwide. Meanwhile, Enterobius vermicularis has been suggested as one of the probable causes of appendicitis. In this study, the morphological characteristics of the remnant pinworms and pathologic changes were explored in old-archived FFPE tissues of appendectomies. Moreover, we provide the first molecular identification, genetic, and haplotype variation of this nematode from the old-archived FFPE tissue section of appendectomy using the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Seventeen FFPE appendectomies with E. vermicularis infection, stored over 12-22 years, were collected from two different geographical areas of Iran. In the histopathological examination, tissue changes were observed in thirteen cases (76.4%) and inflammation in four blocks (23.5%). After DNA extraction, the cox1 gene was amplified in twelve (70.6%) cases using the nested polymerase chain reaction (PCR). Phylogenetic analysis and a median-joining network of 78 available cox1 sequences of E. vermicularis revealed 59 haplotypes. We identified five haplotypes that fell into type B. All Haplotypes are novel except for two haplotypes, Hap32 and Hap37, identical to E. vermicularis sequences from Iran, Greece, and Germany. The ranges of diversity distance and haplotype diversity within the isolates were 0-1.9% and HD:0.643-0.667, subsequently. Overall, the absence of inflammation or even tissue changes in some sections can suggest the possible non-inflammatory role of E. vermicularis in appendicitis. Although FFPE material suffers from PCR inhibition, we could successfully use nested PCR to characterize E. vermicularis in old-archived appendectomy blocks and suggest this method as a complementary diagnosis technique in pathology. While the predominant type was B in the Middle East and Europe, further studies on a larger sample size from different geographical regions could probably confirm the results obtained in the present study.

First Evidence for Colonizing of Acanthamoeba T4 Genotype in Urinary Tracts of Patients with Recurrent Urinary Tract Infections.

BACKGROUND: Limited evidence about the presence of Acanthamoeba spp. in urine specimens collected from urinary catheters of the patients in the intensive care units persuaded our study. No evidence has been found about colonizing of Acanthamoeba spp., in urinary tracts of patients with recurrent urinary tract infections (UTIs) yet. METHODS: In this study, 50 urine samples were collected from patients presenting with recurrent UTI. The type of bacteria causing UTI was determined by using bacteriological tests. To cultivate Acanthamoeba spp., in a sterile condition, 10 mL of urine was centrifuged and the sediment was cultivated on non-nutrient agar. Genotypes were determined by sequencing the DF3 region of the 18S rRNA gene. RESULTS: The bacteriological test findings on the urine samples of the UTI patients (n = 30) demonstrated that those were found to be positive for Escherichia coli (n = 17), Staphylococcus aureus (n = 6), Pseudomonas aeruginosa (n = 4) and Klebsiella spp. (n = 3) respectively. Moreover, a total of 50 urine samples was examined; 6 (6/50; 12%) and 11 (11/50; 22%) were positive by using culture and the PCR test for Acanthamoeba spp., respectively. Sequencing analysis showed all isolates were T4 genotype. CONCLUSIONS: Our data showed that the high relative prevalence of Acanthamoeba T4 genotype spp., in the urine of recurrent UTI patients. As well as, providing the first evidence for colonizing of the Acanthamoeba in the urinary tracts of patients with recurrent UTIs. These findings, warrant further investigation among those patients to fully appraise the role of Acanthamoeba spp., as possible latent carriers for resistant bacteria and biofilm formation in the future.

Management of refractory Acanthamoeba keratitis, two cases.

Acanthamoeba keratitis is a serious infection of the eye that can result in permanent visual impairment or blindness, caused by free-living amoebae of the genus Acanthamoeba. Early diagnosis is necessary for effective treatment of Acanthamoeba keratitis. Acanthamoeba is abundant in nature and can be found in water, soil, and air. Acanthamoeba keratitis is usually diagnosed by culture from a scraping of the eye or by confocal microscopy. In this paper, two complicated Acanthamoeba keratitis cases are reported.

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.

Reviewing the Effects of Miltefosine and Suggesting It for the Treatment of Coronavirus Disease (COVID-19).

OBJECTIVE: Miltefosine is an anti-cancer drug used to treat leishmaniasis and deadly opportunistic free-living amoeba and other deadly pathogenic microorganisms. Several studies have demonstrated its antiviral effect. In this study, we discuss the effectiveness of this drug on pathogenic microorganisms, and according to the functional system of the medicine, we present this drug as a therapeutic proposal to treat Coronavirus disease (COVID-19). METHODS: A literature search was conducted in electronic databases, including Pubmed, Science Direct, Elsevier, and Google Scholar, and articles published from 2006 to 2020 (the last decade) were selected. The search keywords included Miltefosine, microorganism, pathogen, and treatment. RESULTS: The studies indicated that Miltefosine had therapeutic effects on leishmaniasis and deadly opportunistic free-living amoeba and other deadly pathogenic microorganisms. Several studies have proven its antiviral effect. CONCLUSION: Owing to the beneficial effects of this drug on pathogenic and deadly microorganisms and antiviral effects, and due to the epidemic of Coronavirus and the lack of effective treatment and vaccine, this drug is recommended as one of the treatment options for this disease.

Growth comparison of Acanthamoeba genotypes T3 and T4 in several culture media.

Acanthamoeba causes severe diseases such as Granulomatous Amebic Encephalitis (GAE) and Acanthamoeba keratitis (AK). Improving the culture media classically used for this amoeba could help to identify it quickly and facilitate its study as a biological model. The purpose of this study was to compare the growth of two Acanthamoeba genotypes (T3 and T4) in several culture media. Acanthamoeba griffini (T3 genotype) and Acanthamoeba castellanii (T4 genotype) were cultured in PYG, TSY, TYI-S-33, RPMI, and RPMI-FBS medium. The number of amoebas grown in different culture media was counted and compared to each other for 14 days. Findings in this research revealed the highest growth in RPMI-FBS medium. For this reason, we can recommend this culture medium to promote the growth of Acanthamoeba in its biological studies.

Isolation and identification of free-living amoeba from the hot springs and beaches of the Caspian Sea.

Free-living amoeba (FLA) such as Acanthamoeba, Naegleria, Balamuthia, and Vermamoeba have been identified from both natural and human-made environments such as Hot springs and spa. Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), while Acanthamoeba and Balamuthia cause chronic granulomatous encephalitis. Acanthamoeba also can cause cutaneous lesions and Amoebic Keratitis (AK) that is associated with contact lens use or corneal trauma. FLA are known to serve as host of and vehicles for diverse intracellular organisms. This study aimed was to identify the presence of FLA in the hot springs and beaches of the Caspian Sea in Ramsar tourist town located in the northern part of Iran. Water samples were collected in sterile bottles and were transferred to the laboratory. One litre of each sample passed through the nitrocellulose membrane filter. Each filter insert was then placed in non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Positive samples were analyzed by morphological keys and Polymerase chain reaction (PCR) using 18S rDNA gene and ITS region to identify amoeba isolates. A total of 81 water sampled were tasted. After identified using the morphological key and PCR assay, 54 (66.6%) of the samples were positive for FLA. Ten of the samples were identified as Acanthamoeba (belong to T3, T4, and T5 genotypes), three as Vermamoeba vermiformis, four as Naegleria (3 N.australiensis and 1 N.grubery). Only one sample was positive Vahlkampfia. The presence of thermotolerant FLA in the Hot springs and beaches of the Caspian Sea as places for recreational purposes or wellness may be a potential health risk.

First Molecular Evidences of Acanthamoeba T3, T4 and T5 Genotypes in Hemodialysis Units in Iran.

BACKGROUND: Acanthamoeba is a genus of the free-living amoeba that is widespread in the environment and is a causative agent of opportunistic infections in human. This study aimed to investigate the existence and genotyping of Acanthamoeba species in hemodialysis units in Iran. METHODS: In the present study, forty water samples of hydraulic systems and twenty dust samples were collected from two hemodialysis units in Mazandaran Province, northern Iran. The samples were cultivated on non-nutrient agar and genotyping was performed by targeting the 18S rRNA gene. RESULTS: Both morphology and molecular analyses showed that 17.5% (7/40) of water samples and 50% (10/20) of dust samples were positive for Acanthamoeba spp. The sequencing analysis of these isolates was found to be T3, T4 and T5 genotypes. DISCUSSION: To the best of our knowledge, this is the first investigation to identify of Acanthamoeba species in hydraulic system of hemodialysis units in Iran. High contamination of hemodialysis units with virulent T4 genotype of Acanthamoeba may poses a risk for biofilm formation. Our results support urgent need to improve filtration methods in dialysis units and monitoring hemodialysis patients for Acanthamoeba infections.

An Experimental Model of Primary Amoebic Meningoence phalitis Due to Naegleria australiensis in Iran.

BACKGROUND: The main aim of the present research was to develop the experimental meningo encephalitis due to Naegleria australiensis isolated from geothermal water sources in mice model, November 2017 in Iran. METHODS: Naegleria australiensis was isolated from geothermal water sources in northern Iran. The number of amoebae was adjusted to be 1×104/ml amoebae. The experimental infection was done using 3 wk old male (BALB/c) mice. Seven animals were used for experimental amebic infection and one animal was selected for the control. Intranasal (IN) and intracerebral (IC) inoculation of amoebae were done. The mice were then monitored on daily observation and as soon as they present any brain involvement they sacrificed. The brain of all animals was then dislocated and passaged in non-nutrient agar. RESULTS: One mouse out of seven infected mice were showed clinical symptoms of meningoencephalitis. Within few hours of culture of the brain, many vegetative forms of amoebae were detected in plate culture. The other infected animals and control mice showed no clinical symptoms until day 14. After 14 d all the animals sacrificed. The culture was negative up to one month. CONCLUSION: The lack of brain involvement of other animals in the present study could be due to animal immune system or it may be possible that the amoebae did not reach to olfactory bulb of nostrils.

Distribution of Acanthamoeba Genotypes Isolated from Recreational and Therapeutic Geothermal Water Sources in Southwestern Iran.

A comprehensive survey was conducted along 10 km of geothermal rivers in southwestern Iran. A total of 40 water samples were tested for the presence of Acanthamoeba spp., and genotypes were determined by targeting the diagnostic fragment 3 region of the 18S rRNA gene. The pathogenic potential of all positive isolates was also identified using tolerance ability test. High occurrences of Acanthamoeba (50%) were detected in the sampling areas. Based on sequencing analysis, isolates belonging to T4 (93.7%) and T2 (6.25%) genotypes were reported. Thermo- and osmotolerance tests revealed that five strains are highly pathogenic. Since every collection site of this study was associated with high human activity, posting of warning signs, monitoring of recreational water sources, and awareness of high-risk people are of utmost importance. To the best of our knowledge, the present research is the first to report T2 genotype from geothermal water sources in Iran.