Yeast species in the respiratory samples of COVID-19 patients; molecular tracking of Candida auris.

Introduction: Although the existence of Candida species in the respiratory tract is often considered commensal, it is crucial to recognize the significance of Candida colonization in immunocompromised or COVID-19 patients. The emergence of Candida auris as an emerging pathogen further emphasizes the importance of monitoring yeast infection/colonization, particularly in COVID-19 patients. Methods: In this study, respiratory samples mainly from COVID-19 patients, primarily those suspected of having a fungal infection, were cultured on Sabouraud dextrose agar plates and the yeast colonies were identified using a two-step multiplex PCR method. The samples suspected of C. auris underwent specific nested PCR followed by sequence analysis. Results: A total of 199 respiratory samples were collected from 73 women and 126 men, ranging in age from 1.6 to 88 years. Among the patients, 141 had COVID-19, 32 had cancer, 5 were hospitalized in ICU, 2 had chronic obstructive pulmonary disease)COPD(, and others were patients with combination diseases. From these samples, a total of 334 yeast strains were identified. C. albicans (n=132, 39.52%) was the most common species, followed by C. tropicalis (n=67, 20%), C. glabrata (n=56, 16.76%), C. krusei (n=18, 5.4%), C. parapsilosis (n=17, 5.08%), Saccharomyces cerevisiae (n=10, 3%), C. kefyr (n=9, 2.6%), C. dubliniensis (n=7, 2.1%), C. lusitaniae (n=5, 1.5%), C. auris (n=3, 0.9%), C. guilliermondii (n=2, 0.6%), C. rugosa (n=1, 0.3%), C. intermedia (n=1, 0.3%), and Trichosporon spp. (n=1, 0.3%). C. auris was detected in a patient in ICU and two COVID-19 patients. While its presence was confirmed through sequence analysis, our extensive efforts to isolate C. auris were unsuccessful. Conclusion: While C. albicans colonization remains prevalent, our study found no evidence of Candida lung infection. Since the role of Candida colonization in airway secretions remains ambiguous due to limited research, further studies are imperative to shed light on this matter.

Satureja bachtiarica Induces Cancer Cell Death in Breast and Glioblastoma Cancer in 2D/3D Models and Suppresses Breast Cancer Stem Cells.

Overcoming drug resistance and specifically targeting cancer stem cells (CSCs) are critical challenges in improving cancer therapy. Nowadays, the use of novel and native medicinal plants can provide new sources for further investigations for this purpose. The aim of this study was to assess the potential of S. bachtiarica, an endemic plant with diverse medicinal applications, in suppressing and targeting cancer and cancer stem cells in glioblastoma and breast cancer. The effect of S. bachtiarica on viability, migration, invasion, and clonogenic potential of MDAMB-231 and U87-MG cells was assessed in both two- and three-dimensional cell culture models. Additionally, we evaluated its effects on the self-renewal capacity of mammospheres. The experimental outcomes indicated that S. bachtiarica decreased the viability and growth rate of cells and spheroids by inducing apoptosis and inhibited colony formation, migration, and invasion of cells and spheroids. Additionally, colony and sphere-forming ability, as well as the expression of genes associated with EMT and stemness were reduced in mammospheres treated with S. bachtiarica. In conclusion, this study provided valuable insights into the anti-cancer effects of S. bachtiarica, particularly in relation to breast CSCs. Therefore, S. bachtiarica may be a potential adjuvant for the treatment of cancer.

Invasive Fusarium rhinosinusitis in COVID-19 patients: report of three cases with successful management.

Invasive fungal rhinosinusitis (IFRS) is a life-threatening infection that can occur in immunocompromised patients, including those with COVID-19. Although Mucorales and Aspergillus species are the most common causes of IFRS, infections caused by other fungi such as Fusarium are rare. In this report, we present three cases of proven rhinosinusitis fusariosis that occurred during or after COVID-19 infection. The diagnosis was confirmed through microscopy, pathology, and culture, and species identification of the isolates was performed by DNA sequencing the entire ITS1-5.8 rRNA-ITS2 region and translation elongation factor 1-alpha (TEF-1α). Antifungal susceptibility testing was conducted according to CLSI guidelines. The causative agents were identified as Fusarium proliferatum, F. oxysporum + Aspergillus flavus, and F. solani/falciforme. Treatment involved the administration of antifungal medication and endoscopic sinus surgery to remove the affected mucosa, leading to the successful resolution of the infections. However, one patient experienced a recurrence of IFRS caused by A. flavus 15 months later. Early diagnosis and timely medical and surgical treatment are crucial in reducing mortality rates associated with invasive fusariosis. Additionally, the cautious use of corticosteroids in COVID-19 patients is highly recommended.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

BACKGROUND: Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. METHODS: To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 µl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. RESULTS: Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. CONCLUSIONS: Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.

Identification of Polyphenolic Compounds Responsible for Antioxidant, Anti-Candida Activities and Nutritional Properties in Different Pistachio (Pistacia vera L.) Hull Cultivars.

The use of by-products from the agri-food industry is a promising approach for production of value-added, polyphenol-rich dietary supplements or natural pharmaceutical preparations. During pistachio nut processing, a great amount of husk is removed, leaving large biomass for potential re-use. The present study compares antiglycative, antioxidant, and antifungal activities as well as nutritional values of 12 genotypes belonging to four pistachio cultivars. Antioxidant activity was measured using DPPH and ABTS assays. Antiglycative activity was evaluated as inhibition of advanced glycation end product (AGE) formation in the bovine serum albumin/methylglyoxal model. HPLC analysis was performed to determine the major phenolic compounds. Cyanidin-3-O-galactoside (120.81-181.94 mg/100 g DW), gallic acid (27.89-45.25), catechin (7.2-11.01), and eriodictyol-7-O-glucoside (7.23-16.02) were the major components. Among genotypes, the highest total flavonol content (14.8 mg quercetin equivalents/g DW) and total phenolic content (262 mg tannic acid equivalent/g DW) were in KAL1 (Kaleghouchi) and FAN2 (Fandoghi), respectively. The highest antioxidant (EC50 = 375 µg/mL) and anti-glycative activities were obtained for Fan1. Furthermore, potent inhibitory activity against Candida species was recorded with MIC values of 3.12-12.5 µg/mL. The oil content ranged from 5.4% in Fan2 to 7.6% in Akb1. The nutritional parameters of the tested cultivars were highly variable: crude protein (9.8-15.8%), ADF (acid detergent fiber 11.9-18.2%), NDF (neutral detergent fiber, 14.8-25.6%), and condensed tannins (1.74-2.86%). Finally, cyanidin-3-O-galactoside was considered an effective compound responsible for antioxidant and anti-glycative activities.

Microbial and clinical epidemiology of invasive fungal rhinosinusitis in hospitalized COVID-19 patients, the divergent causative agents.

Since COVID-19 spread worldwide, invasive fungal rhinosinusitis (IFRS) has emerged in immunocompromised patients as a new clinical challenge. In this study, clinical specimens of 89 COVID-19 patients who presented clinical and radiological evidence suggestive of IFRS were examined by direct microscopy, histopathology, and culture, and the isolated colonies were identified through DNA sequence analysis. Fungal elements were microscopically observed in 84.27% of the patients. Males (53.9%) and patients over 40 (95.5%) were more commonly affected than others. Headache (94.4%) and retro-orbital pain (87.6%) were the most common symptoms, followed by ptosis/proptosis/eyelid swelling (52.8%), and 74 patients underwent surgery and debridement. The most common predisposing factors were steroid therapy (n = 83, 93.3%), diabetes mellitus (n = 63, 70.8%), and hypertension (n = 42, 47.2%). The culture was positive for 60.67% of the confirmed cases, and Mucorales were the most prevalent (48.14%) causative fungal agents. Different species of Aspergillus (29.63%) and Fusarium (3.7%) and a mix of two filamentous fungi (16.67%) were other causative agents. For 21 patients, no growth was seen in culture despite a positive result on microscopic examinations. In PCR-sequencing of 53 isolates, divergent fungal taxons, including 8 genera and 17 species, were identified as followed: Rhizopus oryzae (n = 22), Aspergillus flavus (n = 10), A. fumigatus (n = 4), A. niger (n = 3), R. microsporus (n = 2), Mucor circinelloides, Lichtheimia ramosa, Apophysomyces variabilis, A. tubingensis, A. alliaceus, A. nidulans, A. calidoustus, Fusarium fujikuroi/proliferatum, F. oxysporum, F. solani, Lomentospora prolificans, and Candida albicans (each n = 1). In conclusion, a diverse set of species involved in COVID-19-associated IFRS was observed in this study. Our data encourage specialist physicians to consider the possibility of involving various species in IFRS in immunocompromised and COVID-19 patients. In light of utilizing molecular identification approaches, the current knowledge of microbial epidemiology of invasive fungal infections, especially IFRS, may change dramatically.

Invasive fungal rhinosinusitis (IFRS) may infect people with diabetes, cancer, or COVID-19. In this study, various types of fungi were identified from COVID-19-associated-IFRS, encouraging physicians to consider specific treatments.

Diagnosis of echinococcosis by detecting circulating cell-free DNA and miRNA.

INTRODUCTION: Diagnosis of echinococcosis is difficult and usually performed based on clinical findings, imaging, and serological test. However, all of them have limitations, especially in follow-up approaches. AREAS COVERED: Detection of cell-free DNA (cfDNA) and micro-RNA (miRNA) is currently a hot topic for diagnosis of echinococcosis diseases. For detecting cell-free DNA in echinococcosis patient's samples such as sera, some techniques are based on next-generation sequencing (NGS), DNA-deep sequencing, some are based on PCR-based methods, and a few works related to the detection of miRNA for the diagnosis of human echinococcosis. EXPERT OPINION: In the detection of cell-free DNA in echinococcosis patient' samples, NGS and DNA-deep sequencing have shown high level of sensitivity, but are not suitable for routine clinical examination as they are expensive and inaccessible in the majority of endemic areas. However, PCR-based methods have shown a sensitivity of about 20-25%. To improve the sensitivity of these tests, improving the DNA extraction method, designing appropriate primers for detecting short-length fragments of circulating DNA, using a higher volume of a serum sample, and application of more sensitive PCR methods are recommended. In the field of miRNA detection, further works are recommended.

Species distribution and susceptibility profiles of oral candidiasis in hematological malignancy and solid tumor patients.

Oral colonization and infection by Candida species are common in cancer patients receiving chemoradiotherapy, which has significantly increased in recent years. This study aimed to evaluate the frequency, distribution, and antifungal susceptibility profiles of Candida species isolates in patients with hematological malignancy and solid tumors. This study was conducted on a total of 45 cancer patients undergoing treatment with concurrent chemoradiotherapy within 2019-2020. The identification of Candida species was accomplished based on conventional examination and molecular assays. The minimum inhibitory concentrations were determined based on the guidelines of Clinical and Laboratory Standards Institute. The highest prevalence rates of oral candidiasis were observed in patients with chronic lymphoid leukemia (24.4%) and lymphoma (20%). The majority of the patients had oral candidiasis caused by non-albicans Candida species (64.4%). The results of the multiplex PCR for the identification of Candida glabrata, Candida nivariensis, Candida bracarensis, and species-specific Candida parapsilosis complex showed that all isolate amplification products at 397 bp and 171 bp were related to C. glabrata and C. parapsilosis, respectively. There was a significant difference in the Candida species distribution between the hematological malignancies and solid tumors patients. The results of MIC showed that clotrimazole, voriconazole, and caspofungin were the most effective antifungal drugs against oral non-Candida albicans isolates. An understanding of the epidemiology of oral candidiasis among hematological malignancies and solid tumors patients is currently imperative to guide optimal empirical treatment strategies for affected patients.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

Fatal Invasive Pulmonary Aspergillosis in COVID-19 Patient with Acute Myeloid Leukemia in Iran.

Although patients with severe immunodeficiency and hematological malignancies has been considered at highest risk for invasive fungal infection, patients with severe pneumonia due to influenza, and severe acute respiratory syndrome coronavirus (SARS-CoV) are also at a higher risk of developing invasive pulmonary aspergillosis (IPA). Recently, reports of IPA have also emerged among SARS-CoV-2 infected patients admitted to intensive care units (ICUs). Here, we report a fatal case of probable IPA in an acute myeloid leukemia patient co-infected with SARS-CoV-2 and complicated by acute respiratory distress syndrome (ARDS). Probable IPA is supported by multiple pulmonary nodules with ground glass opacities which indicate halo sign and positive serum galactomannan results. Screening studies are needed to evaluate the prevalence of IPA in immunocompromised patients infected with SARS-CoV-2. Consequently, testing for the presence of Aspergillus in lower respiratory secretions and galactomannan in consecutive serum samples of COVID-19 patients with timely and targeted antifungal therapy based on early clinical suspicion of IPA are highly recommended.

The first rare and fatal case of invasive aspergillosis of spinal cord due to Aspergillus nidulans in an Iranian child with chronic granulomatosis disease: review of literature.

BACKGROUND AND PURPOSE: Invasive aspergillosis (IA) of the central nervous system (CNS) is a devastating complication which is rarely reported in immunocompromised children. In this case presentation, we reported a rare and fatal IA with spinal cord involvement in a 10-year-old child with X-linked chronic granulomatosis disease (CGD). CASE REPORT: The child had a previous history of pulmonary tuberculosis. A cervical spine X-ray revealed the involvement of cervical vertebrae (T4/T5) and ribs causing spinal cord compression and epidural abscess. The patient underwent a decompressive laminectomy and mass removal. The histopathology and culture results suggested IA. Despite the aggressive and prolonged therapy, he died within one year. Aspergillus nidulans was identified as the causative agent based on morphological and molecular studies. CONCLUSION: This synopsis represents the aggressive behavior of infection caused by A. nidulans in the CGD patient.

In Vitro Activity of Amphotericin B in Combination with Statins against Clinical and Environmental Rhizopus oryzae Strains.

BACKGROUND: Mucormycosis is an acute and invasive fungal infection with a high mortality rate. Mucorales are less sensitive than other types of fungi to most antifungal agents. Amphotericin B (AMB) is one treatment option for this infection, but in recent studies, the antifungal activity of statins against Mucorales was shown. Therefore, therapy that combines AMB with these agents may have better effects in management of patients with mucormycosis. We evaluated the in vitro activity of AMB alone and in combination with statins, against Mucorales. METHODS: Susceptibility profiles of AMB alone and in combination with two statins, atorvastatin (ATO) and lovastatin (LOV) determined against clinical (n: 15) and environmental (n: 5) Rhizopus oryzae isolates, obtained between Jan 2009 and Oct 2016 from patients with uncontrolled diabetes mellitus and cancer referred to the Department of Parasitology and Medical Mycology of Tehran University of Medical Sciences, Tehran, Iran. It was performed by microdilution method, based on the Clinical and Laboratory Standard Institute (CLSI) M38-A2 guideline. RESULTS: All clinical and environmental isolates were susceptible to AMB (MIC≤1 µg/mL). The results of the interactions between AMB and the two statins were positive. The AMB-ATO (GM: 0.13 µg/Ml) combination produced greater activity than the AMB-LOV (GM: 0.26 µg/mL) combination. AMB, in combination with ATO and LOV, reacts positively against clinical and environmental R. oryzae isolates. CONCLUSION: This combination strategy may lead to more effective treatment of mucormycosis and fewer side effects using low dose of AMB.

Analysis of nad2 and nad5 enables reliable identification of genotypes G6 and G7 within the species complex Echinococcus granulosus sensu lato.

The larval stages of tapeworms in the species complex Echinococcus granulosus sensu lato cause a zoonotic disease known as cystic echinococcosis (CE). Within this species complex, genotypes G6 and G7 are among the most common genotypes associated with human CE cases worldwide. However, our understanding of ecology, biology and epidemiology of G6 and G7 is still limited. An essential first step towards this goal is correct genotype identification, but distinguishing genotypes G6 and G7 has been challenging. A recent analysis based on complete mitogenome data revealed that the conventional sequencing of the cox1 (366 bp) gene fragment mistakenly classified a subset of G7 samples as G6. On the other hand, sequencing complete mitogenomes is not practical if only genotype or haplogroup identification is needed. Therefore, a simpler and less costly method is required to distinguish genotypes G6 and G7. We compared 93 complete mitogenomes of G6 and G7 from a wide geographical range and demonstrate that a combination of nad2 (714 bp) and nad5 (680 bp) gene fragments would be the best option to distinguish G6 and G7. Moreover, this method allows assignment of G7 samples into haplogroups G7a and G7b. However, due to very high genetic variability of G6 and G7, we suggest to construct a phylogenetic network based on the nad2 and nad5 sequences in order to be absolutely sure in genotype assignment. For this we provide a reference dataset of 93 concatenated nad2 and nad5 sequences (1394 bp in total) containing representatives of G6 and G7 (and haplogroups G7a and G7b), which can be used for the reconstruction of phylogenetic networks.

Distinguishing Echinococcus granulosus sensu stricto genotypes G1 and G3 with confidence: A practical guide.

Cystic echinococcosis (CE), a zoonotic disease caused by tapeworms of the species complex Echinococcus granulosus sensu lato, represents a substantial global health and economic burden. Within this complex, E. granulosus sensu stricto (genotypes G1 and G3) is the most frequent causative agent of human CE. Currently, there is no fully reliable method for assigning samples to genotypes G1 and G3, as the commonly used mitochondrial cox1 and nad1 genes are not sufficiently consistent for the identification and differentiation of these genotypes. Thus, a new genetic assay is required for the accurate assignment of G1 and G3. Here we use a large dataset of near-complete mtDNA sequences (n = 303) to reveal the extent of genetic variation of G1 and G3 on a broad geographical scale and to identify reliable informative positions for G1 and G3. Based on extensive sampling and sequencing data, we developed a new method, that is simple and cost-effective, to designate samples to genotypes G1 and G3. We found that the nad5 is the best gene in mtDNA to differentiate between G1 and G3, and developed new primers for the analysis. Our results also highlight problems related to the commonly used cox1 and nad1. To guarantee consistent identification of G1 and G3, we suggest using the sequencing of the nad5 gene region (680 bp). This region contains six informative positions within a relatively short fragment of the mtDNA, allowing the differentiation of G1 and G3 with confidence. Our method offers clear advantages over the previous ones, providing a significantly more consistent means to distinguish G1 and G3 than the commonly used cox1 and nad1.

The benefits of analysing complete mitochondrial genomes: Deep insights into the phylogeny and population structure of Echinococcus granulosus sensu lato genotypes G6 and G7.

Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of the species complex Echinococcus granulosus sensu lato. Within this complex, genotypes G6 and G7 have been frequently associated with human CE worldwide. Previous studies exploring the genetic variability and phylogeography of genotypes G6 and G7 have been based on relatively short mtDNA sequences, and the resolution of these studies has often been low. Moreover, using short sequences, the distinction between G6 and G7 has in some cases remained challenging. The aim here was to sequence complete mitochondrial genomes (mitogenomes) to obtain deeper insight into the genetic diversity, phylogeny and population structure of genotypes G6 and G7. We sequenced complete mitogenomes of 94 samples collected from 15 different countries worldwide. The results demonstrated that (i) genotypes G6 and G7 can be clearly distinguished when mitogenome sequences are used; (ii) G7 is represented by two major haplogroups, G7a and G7b, the latter being specific to islands of Corsica and Sardinia; (iii) intensive animal trade, but also geographical isolation, have likely had the largest impact on shaping the genetic structure and distribution of genotypes G6 and G7. In addition, we found phylogenetically highly divergent haplotype from Mongolia (Gmon), which had a higher affinity to G6.

Molecular phylogeny based on six nuclear genes suggests that Echinococcus granulosus sensu lato genotypes G6/G7 and G8/G10 can be regarded as two distinct species.

Tapeworms of the species complex of Echinococcus granulosus sensu lato (s. l.) are the cause of a severe zoonotic disease - cystic echinococcosis, which is listed among the most severe parasitic diseases in humans and is prioritized by the World Health Organization. A stable taxonomy of E. granulosus s. l. is essential to the medical and veterinary communities for accurate and effective communication of the role of different species in this complex on human and animal health. E. granulosus s. l. displays high genetic diversity and has been divided into different species and genotypes. Despite several decades of research, the taxonomy of E. granulosus s. l. has remained controversial, especially the species status of genotypes G6-G10. Here the Bayesian phylogeny based on six nuclear loci (7387 bp in total) demonstrated, with very high support, the clustering of G6/G7 and G8/G10 into two separate clades. According to the evolutionary species concept, G6/G7 and G8/G10 can be regarded as two distinct species. Species differentiation can be attributed to the association with distinct host species, largely separate geographical distribution and low level of cross-fertilization. These factors have limited the gene flow between genotypic groups G6/G7 and G8/G10, resulting in the formation of distinct species. We discuss ecological and epidemiological differences that support the validity of these species.

Global phylogeography and genetic diversity of the zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1.

Echinococcus granulosus sensu stricto (s.s.) is the major cause of human cystic echinococcosis worldwide and is listed among the most severe parasitic diseases of humans. To date, numerous studies have investigated the genetic diversity and population structure of E. granulosus s.s. in various geographic regions. However, there has been no global study. Recently, using mitochondrial DNA, it was shown that E. granulosus s.s. G1 and G3 are distinct genotypes, but a larger dataset is required to confirm the distinction of these genotypes. The objectives of this study were to: (i) investigate the distinction of genotypes G1 and G3 using a large global dataset; and (ii) analyse the genetic diversity and phylogeography of genotype G1 on a global scale using near-complete mitogenome sequences. For this study, 222 globally distributed E. granulosus s.s. samples were used, of which 212 belonged to genotype G1 and 10 to G3. Using a total sequence length of 11,682 bp, we inferred phylogenetic networks for three datasets: E. granulosus s.s. (n = 222), G1 (n = 212) and human G1 samples (n = 41). In addition, the Bayesian phylogenetic and phylogeographic analyses were performed. The latter yielded several strongly supported diffusion routes of genotype G1 originating from Turkey, Tunisia and Argentina. We conclude that: (i) using a considerably larger dataset than employed previously, E. granulosus s.s. G1 and G3 are indeed distinct mitochondrial genotypes; (ii) the genetic diversity of E. granulosus s.s. G1 is high globally, with lower values in South America; and (iii) the complex phylogeographic patterns emerging from the phylogenetic and geographic analyses suggest that the current distribution of genotype G1 has been shaped by intensive animal trade.

Genetic diversity and phylogeography of the elusive, but epidemiologically important Echinococcus granulosus sensu stricto genotype G3.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

Multilocus sequence analysis of Echinococcus granulosus strains isolated from humans and animals in Iran.

Echinococcus granulosus is now considered a complex consisting of at least four species and ten genotypes. Different molecular targets have been described for molecular characterization of E. granulosus; however, in almost all studies only one or two of the targets have been used, and only limited data is available on the utilization of multiple loci. Therefore, we investigated the genetic diversity among 64 strains isolated from 138 cyst specimens of human and animal isolates, using a set of nuclear and mitochondrial genes; i.e., cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunit 1 (nad1), ATPase subunit 6 (atp6), 12S rRNA (12S), and Actin II (act II). In comparison to the use of molecular reference targets (nad1 + cox1), using singular target (act II or 12S or atp6) yielded lower discriminatory power. Act II and 12S genes could accurately discriminate the G6 genotype, but they were not able to differentiate between G1 and G3 genotypes. As the G1 and G3 genotypes belong to the E. granulosus sensu stricto, low intra-species variation was observed for act II and 12S. The atp6 gene could identify the G3 genotype but could not differentiate G6 and G1 genotypes. Using concatenated sequence of five genes (cox1 + nad1 + atp6 + 12S + act II), genotypes were identified accurately, and markedly higher resolution was obtained in comparison with the use of reference markers (nad1 + cox1) only. Application of multilocus sequence analysis (MLSA) to large-scale studies could provide valuable epidemiological data to make efficient control and management measures for cystic echinococcosis.

New mitogenome and nuclear evidence on the phylogeny and taxonomy of the highly zoonotic tapeworm Echinococcus granulosus sensu stricto.

Cystic echinococcosis, a zoonotic disease caused by Echinococcus granulosus sensu lato (s. l.), is a significant global public health concern. Echinococcus granulosus s. l. is currently divided into numerous genotypes (G1-G8 and G10) of which G1-G3 are the most frequently implicated genotypes in human infections. Although it has been suggested that G1-G3 could be regarded as a distinct species E. granulosus sensu stricto (s. s.), the evidence to support this is inconclusive. Most importantly, data from nuclear DNA that provide means to investigate the exchange of genetic material between G1-G3 is lacking as none of the published nuclear DNA studies have explicitly included G2 or G3. Moreover, the commonly used relatively short mtDNA sequences, including the complete cox1 gene, have not allowed unequivocal differentiation of genotypes G1-G3. Therefore, significantly longer mtDNA sequences are required to distinguish these genotypes with confidence. The main aim of this study was to evaluate the phylogenetic relations and taxonomy of genotypes G1-G3 using sequences of nearly complete mitogenomes (11,443bp) and three nuclear loci (2984bp). A total of 23 G1-G3 samples were analysed, originating from 5 intermediate host species in 10 countries. The mtDNA data demonstrate that genotypes G1 and G3 are distinct mitochondrial genotypes (separated by 37 mutations), whereas G2 is not a separate genotype or even a monophyletic cluster, but belongs to G3. Nuclear data revealed no genetic separation of G1 and G3, suggesting that these genotypes form a single species due to ongoing gene flow. We conclude that: (a) in the taxonomic sense, genotypes G1 and G3 can be treated as a single species E. granulosus s. s.; (b) genotypes G1 and G3 should be regarded as distinct genotypes only in the context of mitochondrial data; (c) we recommend excluding G2 from the genotype list.