Elham Kazemirad

Acanthamoeba keratitis (AK) is a severe eye infection that can cause severe vision loss, especially in contact lens wearers. Rapid and accurate diagnosis is crucial for achieving favorable outcomes. Traditional methods, such as culture, are slow and have variable sensitivity, while molecular techniques offer improved detection. This study evaluated nested polymerase chain reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene for rapid AK diagnosis, comparing its performance to one-step PCR, culture, and in vivo confocal microscopy (IVCM). This study included 42 suspected AK patients who underwent IVCM and corneal scraping for culture, PCR, and nested PCR. AK diagnosis was confirmed based on clinical findings, risk factors, treatment response, and at least one positive diagnostic test. Out of 42 patients, 27 were positive for Acanthamoeba with at least one of the diagnostic methods. Based on the diagnosis of definite AK, the sensitivity of IVCM, culture, PCR, and nested PCR was 77.78%, 37.04%, 62.96%, and 96.3%, respectively. The specificity and positive predictive values were 100% for all the tests. The negative predictive values of IVCM, culture, PCR, and nested PCR were 71.43%, 46.88%, 60%, and 93.75%, respectively. Nested PCR results displayed the highest agreement with IVCM. In conclusion, nested PCR enhances AK detection compared to conventional methods, improving sensitivity in low corneal scrape quantities. Combining high-sensitivity tests increases diagnostic accuracy, and nested PCR with IVCM is the most effective approach. When IVCM is unavailable, nested PCR serves as a reliable diagnostic tool, especially when culture and one-step PCR yield negative results.

This report describes the case of a 28-year-old woman, who presented with a two-week history of severe ocular pain and photophobia in her right eye. She was a soft contact lens wearer with no history of ocular trauma or systemic illness. Ophthalmic examination revealed conjunctival hyperemia, epithelial irregularities, and a developing ring-shaped stromal infiltrate. In vivo confocal microscopy demonstrated double-walled hyperreflective cysts, and culture followed by PCR and sequencing confirmed the presence of Acanthamoeba T4 genotype. Despite intensive treatment with topical polyhexamethylene biguanide, chlorhexidine, propamidine, and voriconazole, as well as oral miltefosine, the infection progressed, leading to stromal necrosis. The patient underwent two therapeutic penetrating keratoplasties; however, the disease continued to advance, eventually resulting in phthisis bulbi and permanent vision loss. This case highlights the potential for aggressive, clinically refractory Acanthamoeba keratitis caused by the T4 genotype, emphasizing the importance of early diagnosis, vigilant monitoring, and timely surgical intervention in resistant cases.

Background Increasing resistance to antimonial drugs has become a significant challenge in effective treatment in endemic regions of leishmaniasis. Antioxidant defense plays a crucial role in antimony resistance by combating antimonial-induced oxidative stress. Accordingly, we investigated the transcript levels of some antioxidant genes in antimony-sensitive and resistant clinical Leishmania tropica isolates. Methods In the current study, 22 Leishmania tropica isolates from ACL patients who presented responsive or unresponsive to antimony were examined. The susceptibility of parasites against SbV and hydrogen peroxide (H₂O₂) was analyzed. We evaluated the transcript levels of five genes, including cytosolic and mitochondrial tryparedoxin (cTXN, mTXN), cytosolic and mitochondrial tryparedoxin peroxidase (cTXNPx, mTXNPx), and ascorbate peroxidase (APX) in antimony-sensitive and resistant L. tropica clinical isolates. Results The in-vitro susceptibility to SbV in intracellular amastigotes revealed 3.82 times higher IC₅₀ in resistant isolates compared to sensitive ones. The IC₅₀ toward H₂O₂ in resistant isolates was 1.6 times higher than in sensitive ones, positively correlated with SbV IC₅₀ values. The average transcript expression level of cTXNPx, mTXNPx, cTXN, and APX genes significantly increased in resistant isolates by 2.51, 1.69, 2.41, and 2.12-fold compared to sensitive ones. The highest correlation coefficient between the gene expression and SbV IC₅₀ values belonged to the cTXNPx, cTXN, APX, mTXN, and mTXNPx genes, respectively. The average transcript expression level of cytosolic TXN and TXNPx in resistant L. tropica isolates was higher than its mitochondrial counterpart. Conclusions The data presented here revealed a phenotypic heterogeneity in antioxidant gene expression among L. tropica clinical isolates. Overall, the upregulation of genes involved in antioxidant defense could probably contribute to natural antimony resistance in L. tropica clinical isolates.

Background: We aimed to investigate the prevalence of Enterocytozoon bieneusi and the circulating genotypes in wild boar in the western regions of Iran. Methods: Fifty-two fecal samples were collected from wild boars in Lorestan province, Iran. After DNA extraction, the presence of E. bieneusi was evaluated by real-time PCR. A nested PCR targeting the internal transcribed region (ITS) was employed to characterize genotypes. The PCR products were sequenced, and the genetic diversity and relationships among the genotypes were identified using MEGA X and DnaSp (V5) software. Results: Sixteen (30%) samples were positive for E. bieneusi using real-time PCR, and 11 (21%) were positive for nested PCR, which was sequenced. All 11 positive samples were identified as the BEB6 genotype (Group 2). The haplotype diversity was noted to be 0.182, and the nucleotide diversity, calculated using DnaSp, was 0. 00085. Conclusion: The findings highlight the high prevalence of the genotype BEB6 in wild boars. The presence of this genotype suggests the circulation of E. bieneusi between domesticated animals and wild boars in Iran.

Leishmaniasis is a parasitic disease caused by an intracellular protozoan, Leishmania. Various factors, including host immunity and the Leishmania species, influence the manifestation and severity of the disease. Recent investigations have shed light on the potentially significant role of Leishmania RNA virus (LRV) in the clinical prognosis of leishmaniasis. This study aims to investigate the influence of LRV2 + on various pathogenic genes of Leishmania. In this study, 35 Leishmania isolates were obtained from patients diagnosed with cutaneous leishmaniasis (CL). Leishmania species and the presence of LRV2 + were identified with the PCR-RFLP and semi-nested PCR methods, respectively. Additionally, the RNA expression levels of cysteine protease (CP), heat shock protein 70 (HSP70), heat shock protein 83 (HSP83), glycoprotein 63 (GP63), and mannose phosphate isomerase (MPI) were assessed in LRV2 + and LRV2- Leishmania clinical isolates using RT-qPCR. Out of the 35 isolates, 20 were selected from CL patients, all confirmed as Leishmania major. These isolates were divided into two groups, LRV2 + and LRV2-, with 10 isolates in each group. RT-qPCR analysis revealed that HSP83, MPI, and GP63 gene expression levels were statistically upregulated in LRV2 + isolates compared to LRV2- isolates (P < 0.05). Although HSP70 and CP genes showed slight up-regulation in LRV2 + isolates, it was not statistically significant compared to LRV2- isolates. The notable increase in gene expression levels, particularly for GP63, HSP83, and MPI genes, suggests that the presence of LRV2 + may significantly influence the expression of these factors in L. major clinical isolates. Not applicable.

Free-living amoebae (FLA) are ubiquitous protozoa capable of enduring harsh environmental conditions. These microorganisms are commonly found in water, soil, and air and can be transmitted to humans in areas with high human activity. This study aimed to investigate the prevalence of FLA and their associated genotypes/species in five rivers in Tehran province, Iran. A total of 60 water samples were collected from the Jajrud, Kan, Farhzad, Darakeh, and Shadchay rivers. Samples were subjected to filtration and cultivation onto non-nutrient agar. The genera/species of FLA were characterized based on the amplification and sequencing of the specific genetic fragments. Microscopic analysis suggested the presence of trophozoites and cysts of FLA in 18/60 (30%) of samples, of which Acanthamoeba spp., Vermamoeba sp., and Vahlkampfiidae were identified. Sequence analysis showed the presence of the genotypes T11, T4, T3, and T5 in five, five, four, and one isolates, respectively. The molecular analysis of the T4 genotype showed gene flow between the current isolates with previously described sequences. The findings suggest a clear association between environmental and clinical isolates of Acanthamoeba spp. Therefore, scheduled monitoring of environmental waters, particularly in regions with high human activities, is highly recommended.

Cutaneous leishmaniasis (CL) is a tropical disease that can cause chronic lesions and leave life-long scars, leading to social stigmatization and psychological disorders. Using growth factors and immunomodulatory agents that could accelerate wound healing and reduce the scar is highly demanded. Epidermal growth factor (EGF) plays an essential role in wound healing. It stimulates the proliferation of keratocytes and fibroblasts, and promotes re-epithelialization. Here, the effect of EGF in combination with Glucantime and nano-liposomal Amphotericin B (SinaAmpholeish) on the healing process of CL in BALB/c mice was investigated. Seventy-two mice were infected with Leishmania major parasites and randomly divided into eight treatment groups after the appearance of the lesion. The treatment was continued for five weeks, and lesion sizes were measured weekly. Parasite load was determined in the skin biopsies using qPCR. We found that subcutaneous injection of EGF at 4.5 μg/kg, combined with each of the two antileishmanial drugs, significantly reduced the wound size and parasite load; however, EGF at 1.5 μg/kg failed to be effective. Besides, the wound size and parasite loads were significantly lower in the SinaAmfoleish groups compared to the Glucantime groups. Among the treatment groups, EGF 4.5 μg/kg combined with SinaAmpholeish exhibited the most significant reduction in wound size and parasitic load. Our results suggest that EGF can potentiate the wound healing effect of antileishmanial drugs. Further studies are warranted to explore the beneficial effects of combining EGF with antileishmanial drugs in patients with cutaneous leishmaniasis in order to accelerate wound healing and reduce the scar.