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.

Diagnosis of mucormycosis using a simple duplex PCR assay: Analysis of 160 clinical samples from COVID-19 patients.

Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175 bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements.

Early diagnosis of mucormycosis, a severe complication in COVID-19 patients, is critical for reducing the mortality of the infection. In this study, a sensitive and rapid PCR assay to detect all Mucorales and delineate Rhizopus oryzae was developed and assessed to improve the diagnosis of mucormycosis.

Parasites as potential targets for cancer immunotherapy.

Parasites and cancers have some common antigens. Much scientific evidence in the human population, animal models, and in vitro experiments exhibit that parasites have significant anti-cancer effects. The larval stage of the tapeworm Echinococcus granulosus, Toxoplasma gondii, Trypanosoma cruzy, Plasmodium's, and Trichinella spiralis are among the parasites that have been subjects of anti-cancer research in the last decades. Anti-tumor effects of parasites may be due to the direct impact of the parasites per se or indirectly due to the immune response raised against common antigens between malignant cells and parasites. This manuscript reviews the anti-cancer effects of parasites and possible mechanisms of these effects. Options for using parasites or their antigens for cancer treatment in the future have been discussed.

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.

Withdrawn: Effect of Ursodeoxycholic Acid, Lawsonia Inermis and Achil-lea Millefolium Extracts on Hydatid Cyst Protoscolices

The article has been withdrawn on the request of the authors and the editor of the journal Infectious Disorders - Drug Targets.Bentham Science apologizes to the readers of the journal for any inconvenience this may have caused. Bentham Science Disclaimer: It is a condition of publication that manuscripts submitted to this journal have not been published and will not be simultaneous-ly submitted or published elsewhere. Furthermore, any data, illustration, structure or table that has been published elsewhere must be reported, and copyright permission for reproduction must be obtained. Plagiarism is strictly forbidden, and by submit-ting the article for publication the authors agree that the publishers have the legal right to take appropriate action against the authors, if plagiarism or fabricated information is discovered. By submitting a manuscript the authors agree that the copyright of their article is transferred to the publishers, if and when the article is accepted for publication.

Immunological diagnosis of human hydatid cyst using Western immunoblotting technique.

BACKGROUND: Echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms Echinococcus granulosus. Diagnosis of the disease relies on imaging techniques, but the techniques are not able to differentiate the cyst from benign or malignant tumors; hence, appropriate serologic methods are required for the differential diagnosis of the infection. MATERIALS AND METHODS: In this investigation, different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using Western immunoblotting technique. Considering results of surgery as gold standard, sensitivity and specificity of Western blotting was estimated. RESULTS: Sera of 29, 26, and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid (HCF), protoscolex crude antigen, and cyst wall crude antigen, respectively. However, none of the normal human sera reacted with those specific bands. CONCLUSION: A 20 kDa band of sheep HCF is an appropriate antigen for serodiagnosis of hydatid cyst infection.

Use of restriction fragment length polymorphism to identify Candida species, related to onychomycosis.

BACKGROUND: Onychomycosis is one of the most common clinical forms of fungal infections due to both filamentous fungi and yeasts. The genus of Candida is one of the most prominent causes of onychomycosis in all around the world. Although Candida albicans is still the most frequent cause of nail infections, use of broad-spectrum antifungal agents has led to a shift in the etiology of C. albicans to non-albicans species. The aim of the present study is rapid and precise identification of candida species isolated from nail infection by using of PCR-RFLP technique. MATERIALS AND METHODS: A total of 360 clinical yeast strains were collected from nail infections in Iran. Genomic DNA was extracted using FTA(;) cards. ITS1-5.8SrDNA-ITS2 region was amplified using universal primers and subsequently products were digested with the restriction enzyme MspI. For identification of newly described species (C. parapsilosis complex), the SADH gene was amplified, followed by digestion with Nla III restriction enzyme. RESULTS: Candida albicans was the most commonly isolated species (41.1%), followed by C. parapsilosis (21.4%), C. tropicalis (12.8%), C. kefyr (9.4%), C. krusei (5.5%), C. orthopsilosis (4.1%), C. glabrata (2.8%), C. guilliermondii (1.4%), C. rugosa (0.8%), and C. lusitaniae (0.5%). Patients in the age groups of 51-60 and 81-90 years had the highest and lowest distribution of positive specimens, respectively. CONCLUSION: Rapid and precise identification of Candida species from clinical specimens lead to appropriate therapeutic plans.