An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants.

As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, BA.2, BA.3, BA.4, and BA.5. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) that will be able to distinguish the basic sub-variants of Omicron in a rapid and specific way. Full genome sequences of Omicron strains with high frequency and wide geographical distribution were retrieved by the NCBI Virus and ENA databases. These sequences were compared to each other in order to locate single nucleotide polymorphisms common to all strains of the same sub-variant. These polymorphisms should also be capable of distinguishing Omicron sub-variants not only from each other but from previously circulating variants of SARS-CoV-2 as well. Thus, specific primers targeting characteristic polymorphisms of the four Omicron main branches BA.1, BA.2, BA.4, and BA.5 were designed according to the principles of the amplification refractory mutation system (ARMS) and with the ability to react under multiplex PCR conditions. According to our results, the ARMS-multiplex PCR could successfully distinguish all Omicron sub-variants that carry the corresponding mutations.

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.

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.

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.

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.

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models.

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies.

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.

Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential for Endoplasmic Reticulum to Outer Mitochondrial Membrane Protein Transport.

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA's ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub-domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria-associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients.

Vancomycin-resistance phenotypes, vancomycin-resistance genes, and resistance to antibiotics of enterococci isolated from food of animal origin.

In the present study, 500 raw beef, pork, and chicken meat samples and 100 pooled egg samples were analyzed for the presence of vancomycin-resistant enterococci, vancomycin-resistance phenotypes, and resistance genes. Of 141 isolates of enterococci, 88 strains of Enterococcus faecium and 53 strains of E. faecalis were identified. The most prevalent species was E. faecium. Resistance to ampicillin (n = 93, 66%), ciprofloxacin (n = 74, 52.5%), erythromycin (n = 73, 51.8%), penicillin (n = 59, 41.8%) and tetracycline (n = 52, 36.9%) was observed, while 53.2% (n = 75) of the isolates were multiresistant and 15.6% (n = 22) were susceptible to all antibiotics. Resistance to vancomycin was exhibited in 34.1% (n = 30) of the E. faecium isolates (n = 88) and 1.9% (n = 1) of the E. faecalis isolates (n = 53) using the disc-diffusion test and the E-test. All isolates were tested for vanA and vanB using real-time polymerase chain reaction (PCR) and multiplex PCR, and for vanC, vanD, vanE, vanG genes using multiplex PCR only. Among E. faecalis isolates, no resistance genes were identified. Among the E. faecium isolates, 28 carried the vanA gene when tested by multiplex PCR and 29 when tested with real-time PCR. No isolate carrying the vanC, vanD, vanE, or vanG genes was identified. Melting-curve analysis of the positive real-time PCR E. faecium isolates showed that 22 isolates carried the vanA gene only, 2 isolates the vanB2,3 genes only, and seven isolates carried both the vanA and vanB2,3 genes. Enterococci should be considered a significant zoonotic pathogen and a possible reservoir of genes encoding resistance potentially transferred to other bacterial species.

HSP70 polymorphisms in first psychotic episode drug-naïve schizophrenic patients.

AIMS: The aim of this study is to investigate differences in HSPA8 polymorphisms between first-episode psychotic (FEP) schizophrenic patients and healthy participants after adjustment for temperamental personality traits. MAIN METHODS: This study included fifty drug-naive schizophrenic patients with an FEP and fifty healthy participants who served as controls. Genotyping of HSPA8 polymorphisms was performed in patients and healthy subjects as well. Personality characteristics were assessed using the standardized Greek version of the Alternative Five-Factor Zuckerman-Kuhlman Personality Questionnaire (ZKPQ). KEY FINDINGS: Our results showed that FEP patients presented a polymorphism differentiation related to the HSPA8 gene (rs1136141), and a higher frequency of T carriers compared to healthy controls was observed. The HSP8A polymorphism and the levels of Neuroticism as measured by the Alternative Five-Factor ZKPQ were the variables most closely and independently associated with FEP in multiple logistic regression analysis, and the odds of being assessed with a FEP was 2.8 times greater in T carriers compared to non-carriers. SIGNIFICANCE: Present findings indicate a role of HSP8A in FEP and underline the importance of including personality traits in the study of the factors associated with the development of schizophrenia.

BDNF serum concentrations in first psychotic episode drug-naïve schizophrenic patients: associations with personality and BDNF Val66Met polymorphism.

AIMS: To investigate the relationship among brain derived neurotrophic factor (BDNF) serum concentrations, BDNF Val66Met polymorphism and personality profile in drug-naïve schizophrenic patients with first-episode psychosis (FEP) and healthy participants. MAIN METHODS: This cross-sectional study included fifty FEP patients and fifty healthy participants who served as controls. To study their personality profile the standardized Greek version of the Alternative Five-Factor Zuckerman-Kuhlman Personality Questionnaire (ZKPQ) was administered. Serum BDNF levels were measured and genotyping of BDNF Val66Met polymorphism was performed in patients and healthy subjects. KEY FINDINGS: FEP patients presented lower BDNF serum concentrations (P=0.002) and higher scores in ZKPQ Neuroticism (P=0.001) and Aggression-Hostility (P=0.002) scales while lower scores in the ZKPQ Sociability scale (P<0.001) than healthy participants. Multivariate analysis revealed that the odds of being assessed with FEP were 0.4 times lower in those with higher BDNF values (P<0.001) and 1.8 times greater in those with higher Neuroticism scores (P<0.001). There were no significant differences with respect to the Val66Met polymorphism between patients and healthy participants. SIGNIFICANCE: Reduced BDNF serum concentrations along with higher Neuroticism scores might be associated with FEP. A complex interplay between BDNF serum concentrations, personality traits, BDNF Val66Met polymorphism, and psychotic symptomatology has been arisen but further investigation is needed to better clarify the observed associations.

Trafficking of UL37 proteins into mitochondrion-associated membranes during permissive human cytomegalovirus infection.

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.

Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion-associated membranes of human cells.

The human cytomegalovirus (HCMV) UL37 exon 1 protein (pUL37x1), also known as vMIA, is the predominant UL37 isoform during permissive infection. pUL37x1 is a potent antiapoptotic protein, which prevents cytochrome c release from mitochondria. The UL37x1 NH(2)-terminal bipartite localization signal, which remains uncleaved, targets UL37 proteins to the endoplasmic reticulum (ER) and then to mitochondria. Based upon our findings, we hypothesized that pUL37x1 traffics from the ER to mitochondria through direct contacts between the two organelles, provided by mitochondrion-associated membranes (MAMs). To facilitate its identification, we cloned and tagged the human phosphatidylserine synthase 1 (huPSS-1) cDNA, whose mouse homologue localizes almost exclusively in the MAM. Using subcellular fractionation of stable HeLa cell transfectants expressing mEGFP-huPSS-1, we found that HCMV pUL37x1 is present in purified microsomes, mitochondria, and MAM fractions. We further examined the trafficking of the full-length UL37 glycoprotein cleavage products, which divergently traffic either through the secretory apparatus or into mitochondria. Surprisingly, pUL37(NH2) and gpUL37(COOH) were both detected in the ER and MAM fraction, even though only pUL37(NH2) is preferentially imported into mitochondria but gpUL37(COOH) is not. To determine the sequences required for MAM importation, we examined pUL37x1 mutants that were partially defective for mitochondrial importation. Deletion mutants of the NH(2)-terminal UL37x1 mitochondrial localization signal were reduced in trafficking into the MAM, indicating partial overlap of MAM and mitochondrial targeting signals. Taken together, these results suggest that HCMV UL37 proteins traffic from the ER into the MAM, where they are sorted into either the secretory pathway or to mitochondrial importation.

Antirestenotic effects of a novel polymer-coated d-24851 eluting stent. Experimental data in a rabbit iliac artery model.

Experimental and clinical data suggest that stents eluting antiproliferative agents can be used for the prevention of in-stent restenosis. Here we investigate in vitro the antiproliferative and apoptotic effect of D-24851 and evaluate the safety and efficacy of D-24851-eluting polymer-coated stents in a rabbit restenosis model (n = 53). Uncoated stents (n = 6), poly (DL: -lactide-co-glycolide) (PLGA)-coated stents (n = 7), and PLGA-coated stents loaded with 0.08 +/- 0.0025 microM (31 +/- 1 mug; low dose; n = 7), 0.55 +/- 0.02 microM (216 +/- 8 mug; high dose; n = 6), and 4.55 +/- 0.1 microM (1774 +/- 39 mug; extreme dose; n = 5) of D-24851 were randomly implanted in New Zealand rabbit right iliac arteries and the animals were sacrificed after 28 days for histomorphometric analysis. For the assessment of endothelial regrowth in 90 days, 12 rabbits were subjected to PLGA-coated (n = 3), low-dose (n = 3), high-dose (n = 3), and extreme-dose (n = 3) stent implantation. In vitro studies revealed that D-24851 exerts its growth inhibitory effects via inhibition of proliferation and induction of apoptosis without increasing the expression of heat shock protein-70, a cytoprotective and antiapoptotic protein. Treatment with low-dose D-24851 stents was associated with a significant reduction in neointimal area and percentage stenosis only compared with bare metal stents (38% [P = 0.029] and 35% [P = 0.003] reduction, respectively). Suboptimal healing, however, was observed in all groups of D-24851-loaded stents in 90 days in comparison with PLGA-coated stents. We conclude that low-dose D-24851-eluting polymer-coated stents significantly inhibit neointimal hyperplasia at 28 days through inhibition of proliferation and enhancement of apoptosis. In view of the suboptimal re-endothelialization, longer-term studies are needed in order to establish whether the inhibition of intimal growth is maintained.