Unusual N Gene Dropout and Ct Value Shift in Commercial Multiplex PCR Assays Caused by Mutated SARS-CoV-2 Strain.

Several SARS-CoV-2 variants have emerged and early detection for monitoring their prevalence is crucial. Many identification strategies have been implemented in cases where sequencing data for confirmation is pending or not available. The presence of B.1.1.318 among prevalent variants was indicated by an unusual amplification pattern in various RT-qPCR commercial assays. Positive samples for SARS-CoV-2, as determined using the Allplex SARS-CoV-2 Assay, the Viasure SARS-CoV-2 Real Time Detection Kit and the GeneFinder COVID-19 Plus RealAmp Kit, presented a delay or failure in the amplification of the N gene, which was further investigated. Whole-genome sequencing was used for variant characterization. The differences between the mean Ct values for amplification of the N gene vs. other genes were calculated for each detection system and found to be at least 14 cycles. Sequencing by WGS revealed that all the N gene dropout samples contained the B.1.1.318 variant. All the isolates harbored three non-synonymous mutations in the N gene, which resulted in four amino acid changes (R203K, G204R, A208G, Met234I). Although caution should be taken when the identification of SARS-CoV-2 variants is based on viral gene amplification failure, such patterns could serve as a basis for rapid and cost-effective screening, functioning as indicators of community circulation of specific variants, requiring subsequent verification via sequencing.

Pathophysiological Aspects of Ocular Toxoplasmosis: Host-parasite Interactions.

PURPOSE: This review aims to present the state of the art to understand the pathophysiology of ocular toxoplasmosis (OT), providing further foundations that would help to improve the future treatment and prognosis of this potentially blinding disease. METHODS: A thorough literature search was performed in PubMed database. An additional search was made in Google Scholar to complete the collected items. RESULTS: Toxoplasma gondii ocular infection is one of the most frequent causes of posterior uveitis. Despite the ocular barriers, the parasite reaches the eye through different mechanisms. Once inside, it remains encysted livelong within the retina, and recurrences cannot be completely avoided. The complexity of host-parasite interactions, leading to the success of this parasite, encompasses host factors such as genetic predisposition, immune status, and age; and parasite factors such as strain diversity, virulence, phylogenetic origin, and geographical distribution. These factors influence the clinical presentation, course, and progression of the disease. Additional elements, such as pregnancy, eating behavior, and environmental, social, and cultural factors may also contribute to this complex balance. CONCLUSIONS: The host-parasite interaction in OT is a complex and multifactorial relationship, with the parasite always on the driving edge of the game. There are still multiple incompletely understood fields to be investigated. Future research would permit further insight into the immune-biology of the parasite and recognition of the host-parasite interplay to improve the diagnostic and management performance.

Ocular toxoplasmosis: a review of the current diagnostic and therapeutic approaches.

PURPOSE: This review aims to summarize the current knowledge concerning the clinical features, diagnostic work-up and therapeutic approach of ocular toxoplasmosis focusing mainly on the postnatally acquired form of the disease. METHODS: A meticulous literature search was performed in the PubMed database. A supplementary search was made in Google Scholar to complete the collected items. RESULTS: Ocular toxoplasmosis is one of the most frequent infectious etiologies of posterior uveitis. It typically presents with retinochoroiditis. Setting an accurate diagnosis depends to a considerable degree on detecting characteristic clinical characteristics. In addition to the evaluation of clinical features, the diagnosis of toxoplasmosis relies at a large degree on serologic testing. The detection of the parasite DNA in the aqueous or vitreous humor can provide evidence for a definitive diagnosis. The current mainstay for the treatment, if necessary, is the use of oral antibiotic with systemic corticosteroids. Recent evidence suggests other therapeutic approaches, such as intravitreal antibiotics can be used. CONCLUSION: Recent developments in the diagnostic and therapeutic approach have contributed to preventing or limiting vision loss of patients suffering from ocular toxoplasmosis. Further studies are required to provide a better understanding of epidemiology, pathogenesis, diagnosis, and treatment with a significant impact on the management of this challenging clinical entity.

Modeling microbial ethanol production by S. aureus, K. pneumoniae, and E. faecalis under aerobic/anaerobic conditions - applicability to laboratory cultures and real postmortem cases.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse's colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols' concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae's models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.

Modeling postmortem ethanol production by C. albicans: Experimental study and multivariate evaluation.

In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms.

Rapid Detection of blaKPC-9 Allele from Clinical Isolates.

The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the blaKPC-2 from the blaKPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several blaKPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions.

Evaluating Infectious, Neoplastic, Immunological, and Degenerative Diseases of the Central Nervous System with Cerebrospinal Fluid-Based Next-Generation Sequencing.

Cerebrospinal fluid (CSF) is a clear and paucicellular fluid that circulates within the ventricular system and the subarachnoid space of the central nervous system (CNS), and diverse CNS disorders can impact its composition, volume, and flow. As conventional CSF testing suffers from suboptimal sensitivity, this review aimed to evaluate the role of next-generation sequencing (NGS) in the work-up of infectious, neoplastic, neuroimmunological, and neurodegenerative CNS diseases. Metagenomic NGS showed improved sensitivity-compared to traditional methods-to detect bacterial, viral, parasitic, and fungal infections, while the overall performance was maximized in some studies when all diagnostic modalities were used. In patients with primary CNS cancer, NGS findings in the CSF were largely concordant with the molecular signatures derived from tissue-based molecular analysis; of interest, additional mutations were identified in the CSF in some glioma studies, reflecting intratumoral heterogeneity. In patients with metastasis to the CNS, NGS facilitated diagnosis, prognosis, therapeutic management, and monitoring, exhibiting higher sensitivity than neuroimaging, cytology, and plasma-based molecular analysis. Although evidence is still rudimentary, NGS could enhance the diagnosis and pathogenetic understanding of multiple sclerosis in addition to Alzheimer and Parkinson disease. To conclude, NGS has shown potential to aid the research, facilitate the diagnostic approach, and improve the management outcomes of all the aforementioned CNS diseases. However, to establish its role in clinical practice, the clinical validity and utility of each NGS protocol should be determined. Lastly, as most evidence has been derived from small and retrospective studies, results from randomized control trials could be of significant value.

Molecular Analysis of Dirofilaria repens Isolates from Eye-Care Patients in Greece.

PURPOSE: Human ocular dirofilariasis is a zoonotic disease caused by several species of filarioid helminths of the genus Dirofilaria. The aim of this study was to further re-examine five preserved specimens previously isolated from patients with ocular dirofilariasis by molecular means. METHODS: Four of the examined helminths had been stored in unbuffered formaldehyde solution for more than eight years; whereas, the fifth helminth was stored in ethanol buffer for more than two years. For the four specimens stored in formaldehyde, different methods of DNA recovery and amplification were applied and investigated for their efficiency in DNA extraction and PCR amplification. However, the DNA extraction and PCR amplification were successful only for the ethanol-preserved helminth. RESULTS: The genetic identification of the ethanol-preserved specimen as Dirofilaria repens (D. repens) and its phylogenetic position based on the analysis of mitochondrial 12S ribosomal RNA, nuclear 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit one sequences are reported in the present paper. To our knowledge, these are the only deposited sequences related to D. repens that have been isolated in Greece. CONCLUSIONS: Routine laboratory diagnosis is based on phenotypic characteristics of the helminthic parasites, but more accurate diagnosis requires molecular identification. Although the specimens preserved in formalin buffers may be a potential source for the enrichment of parasite genome databases, the DNA recovery of such samples is a challenging task.

Onychomycosis in Northwestern Greece Over a 7-Year Period.

Onychomycosis is considered as one of the major public health problems with a global distribution associated with geographic, demographic and environmental factors, underlying comorbidities and immunodeficiency disorders. This study was conducted to investigate the etiological agents of onychomycosis, in Northwestern Greece during a 7-year period. The study population included 1095 outpatients with clinically suspected onychomycosis that presented to the University Hospital of Ioannina, NW Greece (2011-2017). Samples were examined for causative fungi, and mycological identification was established using standard mycological methods. Demographic data of each patient, comorbidities, localization of infection and history of previous fungal infection were collected. Onychomycosis was diagnosed in 317 of the 1095 suspected cases (28.9%) and the most frequently isolated pathogens were yeasts (50.8%) followed by dermatophytes (36.9%) and non-dermatophyte molds (NDMs) (12.3%). Dermatophytes were mostly involved in toenail onychomycosis (90.6%) and more commonly affected males than females (57.3% vs. 42.7%), while the predominantly isolated pathogen was Τrichophyton rubrum (74.4%) followed by Τrichophyton interdigitale (21.4%). Candida albicans was the most prevalent isolated yeast (82%), whereas among the cases with onychomycosis due to NDMs, Aspergillus spp. were isolated as the principal species (59%). Continuous monitoring should be performed in order to identify possible trends and shifts in species isolation rates and to evaluate the impact of onychomycosis among the general population and high-risk groups.

Detection of Cryptosporidium and Giardia in foods of plant origin in North-Western Greece.

Giardia and Cryptosporidium are recognized as leading causes of waterborne and foodborne diarrhoeal disease with worldwide distribution. The study aimed to determine the protozoan contamination of various foods of plant origin. A total of 72 samples from 27 different varieties of fresh vegetables and fruits were collected from supermarkets and open markets in North-Western Greece and were examined using conventional diagnostic methods. Two out of 72 (2.8%) samples were found positive for Cryptosporidium oocysts, while no sample was found to be positive for Giardia cysts. The results show the presence of protozoan contamination in foods of plant origin, which may constitute a potential health hazard.

Nutritional Status and the Influence of the Vegan Diet on the Gut Microbiota and Human Health.

The human gut microbiota is considered a well-known complex ecosystem composed of distinct microbial populations, playing a significant role in most aspects of human health and wellness. Several factors such as infant transitions, dietary habits, age, consumption of probiotics and prebiotics, use of antibiotics, intestinal comorbidities, and even metabolic diseases may continously alter microbiota diversity and function. The study of vegan diet-microbiota interactions is a rapidly evolving field, since plenty of research has been focused on the potential effects of plant-based dietary patterns on the human gut microbiota. It has been reported that well-planned vegan diets and their associated components affect both the bacterial composition and metabolic pathways of gut microbiota. Certain benefits associated with medical disorders but also limitations (including nutritional deficiencies) have been documented. Although the vegan diet may be inadequate in calorific value, it is rich in dietary fiber, polyphenols, and antioxidant vitamins. The aim of the present study was to provide an update of the existing knowledge on nutritional status of vegan diets and the influence of their food components on the human gut microbiota and health.

Impact of enterococcal urinary tract infections in immunocompromised - neoplastic patients.

Infections in immunocompromised-neoplastic patients represent a severe complication. Among bacteria, Enterococcus species constitute a common causative pathogen of urinary tract infections (UTIs), especially among hospitalized patients with or without urinary tract carcinoma, related commonly to urinary tract abnormalities, urinary catheters or prolonged antibiotic treatment. Although enterococci have been considered more commonly as colonization bacteria in the intestine than virulent agents, they are frequently implicated in UTIs. The high incidence of enterococcal UTIs is associated with several risk factors including age, female gender, previous UTI, diabetes, pregnancy, immunosuppression due to cancer development and progression, renal transplantation and spinal cord injury. Clinical manifestations are usually absent or mild in enterococcal UTIs, which may also become an important source for both bacteremia and endocarditis. Over the last years, the prevalence of multidrug resistant enterococci, particularly vancomycin-resistant E. faecium and E. faecalis has significantly risen worldwide, associated with increased morbidity, limited treatment options and increased health-care costs. In this review, the current knowledge on enterococcal UTIs epidemiology and influence in the corresponding immunocompromised patients is highlighted.

Antimicrobial Resistance in Bacterial Pathogens and Detection of Carbapenemases in Klebsiella pneumoniae Isolates from Hospital Wastewater.

During a six-month period (October 2017-March 2018), the prevalence and susceptibility of important pathogenic bacteria isolated from 12 hospital raw sewage samples in North Western Greece was investigated. The samples were analyzed for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, carbapenemase-producing Klebsiella pneumoniae (CKP), and multidrug-resistant Pseudomonas aeruginosa. Antimicrobial susceptibility testing was performed using the agar diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute. The diversity of carbapenemases harboring K. pneumoniae was examined by two phenotyping screening methods (modified Hodge test and combined disk test), a new immunochromatographic rapid assay (RESIST-4 O.K.N.V.) and a polymerase chain reaction (PCR). The results demonstrated the prevalence of MRSA, vancomycin-resistant Staphylococcus aureus (VRSA), VRE, and CKP in the examined hospital raw sewage samples. In addition, the aforementioned methods which are currently used in clinical laboratories for the rapid identification and detection of resistant bacteria and genes, performed sufficiently to provide reliable results in terms of accuracy and efficiency.

An Update on Sexual Transmission of Zika Virus.

Zika virus (ZIKV) is a single-stranded RNA virus belonging to the arthropod-borne flaviviruses (arboviruses) which are mainly transmitted by blood-sucking mosquitoes of the genus Aedes. ZIKV infection has been known to be rather asymptomatic or presented as febrile self-limited disease; however, during the last decade the manifestation of ZIKV infection has been associated with a variety of neuroimmunological disorders including Guillain⁻Barré syndrome, microcephaly and other central nervous system abnormalities. More recently, there is accumulating evidence about sexual transmission of ZIKV, a trait that has never been observed in any other mosquito-borne flavivirus before. This article reviews the latest information regarding the latter and emerging role of ZIKV, focusing on the consequences of ZIKV infection on the male reproductive system and the epidemiology of human-to-human sexual transmission.

Oropouche Fever: A Review.

Oropouche fever is an emerging zoonotic disease caused by Oropouche virus (OROV), an arthropod transmitted Orthobunyavirus circulating in South and Central America. During the last 60 years, more than 30 epidemics and over half a million clinical cases attributed to OROV infection have been reported in Brazil, Peru, Panama, Trinidad and Tobago. OROV fever is considered the second most frequent arboviral febrile disease in Brazil after dengue fever. OROV is transmitted through both urban and sylvatic transmission cycles, with the primary vector in the urban cycle being the anthropophilic biting midge Culicoides paraensis. Currently, there is no evidence of direct human-to-human OROV transmission. OROV fever is usually either undiagnosed due to its mild, self-limited manifestations or misdiagnosed because its clinical characteristics are similar to dengue, chikungunya, Zika and yellow fever, including malaria as well. At present, there is no specific antiviral treatment, and in the absence of a vaccine for effective prophylaxis of human populations in endemic areas, the disease prevention relies solely on vector control strategies and personal protection measures. OROV fever is considered to have the potential to spread across the American continent and under favorable climatic conditions may expand its geographic distribution to other continents. In view of OROV's emergence, increased interest for formerly neglected tropical diseases and within the One Health concept, the existing knowledge and gaps of knowledge on OROV fever are reviewed.

Shelf-life of smoked eel fillets treated with chitosan or thyme oil.

The present study examined the effect of natural antimicrobials: Chitosan, thyme oil and their combination, on the shelf-life of smoked eel fillets stored under vacuum packaging (VP) at 4°C. Based on sensory odor data smoked eel fillets had a shelf-life of 35 (control), 42 (thyme treated and>49 (thyme, chitosan-thyme treated) days. The thiobarbituric acid value (TBA) value of the control eel sample was significantly higher than the chitosan-thyme-treated eel samples. The use of chitosan singly, or in combination with thyme oil reduced lipid oxidation (TBA) of the smoked eel samples. A trimethylamine nitrogen (TMA-N) value of 10mgN/100g, could be suggested as an indication of smoked eel spoilage initiation. Control and treated eel reached total volatile basic nitrogen (TVB-N) values of 13.1-31.5mgN/100g below the maximum permissible level of TVB-N in fish and fishery products. Eel samples reached the value of 7.0logcfu/g (Total Plate Count, TPC) on days 35 (smoked) and 42 (thyme treated), whereas both chitosan and chitosan-thyme treated eel samples never reached this limit value. Results of our study show thyme or chitosan (singly, or in combination) inhibit the growth of mesophilic bacteria and extend the shelf-life of smoked eel.

Viability of and Escherichia coli O157:H7 and Listeria monocytogenes in a delicatessen appetizer (yogurt-based) salad as affected by citrus extract (Citrox©) and storage temperature.

The antimicrobial effect of citrus extract (at 1 mL/kg [C1] and 2 mL/kg [C2]) on naturally occurring microbiota and inoculated pathogens (E. coli O157:H7 and L. monocytogenes at ca. 6 log cfu/g) in the traditional Greek yogurt-based salad Tzatziki stored at 4, 10, or 21 °C, was examined. Lactic acid bacteria (LAB) were high (8.0-8.5 log cfu/g) and varied only minimally for both the control (untreated) and the citrus extract-treated salad samples, whereas the higher citrus extract concentration yielded the lowest yeast populations, irrespective of temperature, during the entire storage period. Populations of inoculated E. coli (6 log cfu/g) declined in both untreated and citrus extract-treated samples from day 0-70, 35, and 15 at 4, 10, and 21 °C, respectively. Citrus extract had a significant effect on the survival of the inoculated E. coli O157:H7, with reductions of 2.8-4.8 log cfu/g in the citrus extract-treated samples at the end of the storage period. Our data show that L. monocytogenes survived in both untreated and citrus extract-treated samples during the entire storage period, irrespective of the storage temperature. The higher concentration of citrus extract had a significant effect on the survival of L. monocytogenes in the treated samples, and reductions of 1.5-3.0 logs were noted on final day 70, 35 and 15 at 4, 10 and 21 °C, respectively. The results of our study demonstrated the potential of citrus extract as a natural compound that can control the growth of food-borne pathogenic bacteria, such as E. coli O157:H7 and L. monocytogenes in Tzatziki, a yogurt-based salad.

Evaluation of Antimicrobial Photodynamic Therapy Using Indocyanine Green and Near-Infrared Diode Laser Against Enterococcus faecalis in Infected Human Root Canals.

OBJECTIVE: Photodynamic therapy (PDT) is considered a promising adjunct to the currently available endodontic disinfection techniques leading to more effective reduction of intracanal bacteria. The present ex vivo study aimed to assess the antimicrobial effect of PDT using indocyanine green (ICG) as photosensitizer and a near-infrared (NIR) diode laser in root canals of human teeth infected with Enterococcus faecalis. MATERIALS AND METHODS: Ninety single-rooted teeth after chemomechanical preparation and sterilization were contaminated with an E. faecalis strain. The specimens were divided, randomly, into eight experimental groups: (1) PDT with ICG and laser (0.5 W output power-medium-energy fluence), (2) PDT with ICG and laser (1 W output power-high-energy fluence), (3) only laser emission, (4) only ICG, (5) 2.5% sodium hypochlorite (NaOCl) as irrigant, (6) 2.5% NaOCl and PDT with ICG and laser, (7) no treatment (positive control), and (8) no bacterial biofilm growth (negative control). The root canal contents were sampled by flushing and the collected washings were plated on an appropriate culture medium, which was incubated for 48 h at 35°C ± 2.0. The colony-forming units (CFUs) were determined to assess the bactericidal effect of the tested experimental combinations. RESULTS: The microbiological tests revealed that PDT groups, regardless of the overall power, showed significant lower mean log10 CFU levels, than groups 3 and 4 (p < 0.001) and similar reduction of viable counts with group 5. The combination treatment (group 6) achieved adequate reduction of log10 CFU levels in the viable counts. However, no significant difference (p > 0.05) was observed between groups 1, 2, 5, and 6 and significant difference was noticed between groups 3, 4, and 5 (p < 0.001). CONCLUSIONS: ICG-mediated PDT activated by an NIR diode laser provided increased disinfection of the root canal system, but the overall benefit in total bacterial elimination should be further investigated.

Antimicrobial Activity of Basil, Oregano, and Thyme Essential Oils.

For centuries, plants have been used for a wide variety of purposes, from treating infectious diseases to food preservation and perfume production. Presently, the increasing resistance of microorganisms to currently used antimicrobials in combination with the appearance of emerging diseases requires the urgent development of new, more effective drugs. Plants, due to the large biological and structural diversity of their components, constitute a unique and renewable source for the discovery of new antibacterial, antifungal, and antiparasitic compounds. In the present paper, the history, composition, and antimicrobial activities of the basil, oregano, and thyme essential oils are reviewed.