Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece.

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).

Impact of combined mycoplasmataceae and HPV co-infection on females with cervical intraepithelial neoplasia and carcinoma.

PURPOSE: The concurrent prevalence investigation of human papillomavirus (HPV), Mycoplasma hominis (Mh) and Ureaplasma urealyticum (Uu) in women in order to estimate the association of co-infection with cervical lesions. METHODS: The study cohort comprised 120 women with no cervical lesions (control group) and 62 women with abnormal cytological findings from the cervix (cervical intraepithelial lesion/neoplasia) as study group. A combination of molecular analyses was implemented. RESULTS: The presence of HPV infection was shown in 52/62 (83.9%) of women with abnormal cytology. Women with cervix cytological findings were shown to have 17.6 times higher risk for Mh and Uu co-infection (p=0.001). HPV and Uu co-infection were detected with a higher prevalence among women with CIN 3 and invasive cancer. CONCLUSION: These findings are consistent with the notion that microbial co-infections may play an important role in persistent inflammation and progression of cervical lesions.

Sunbathing, a possible risk factor of murine typhus infection in Greece.

BACKGROUND: There are few studies about the presence of murine typhus in Greece. Our objective was to conduct a large scale retrospective investigation to determine the clinical and epidemiological features of patients diagnosed with murine typhus in Greece. METHODOLOGY/PRINCIPAL FINDINGS: From 2012 to 2019 serum samples from hospitalized patients and outpatients throughout Greece suspected for murine typhus infection were tested by immunofluorescence assay for Rickettsia typhi. Immunofluorescence positive samples obtained since 2016 were also tested by qPCR targeting R. typhi. Clinical and epidemiological data were retrospectively collected for the patients with confirmed murine typhus. Overall, we tested 5,365 different patients and, in total, 174 patients from all geographic regions of Greece were diagnosed with murine typhus. The most frequently reported sign or symptom was fever (89%), followed by headache (84%) and rash (81%). The classical triad of fever, headache, and rash was present in 72% of patients during their illness. Severe infections with complications including acute renal failure or septic shock were not recorded. The majority of cases (81%) occurred during May-October and peaked in June and September. Most of patients (81%) infected in Athens, recalled that their only activity the last weeks before symptoms onset was swimming on the beach and 59% of them also reported an insect bite while sunbathing. CONCLUSIONS/SIGNIFICANCE: Our results may reflect the reemergence of murine typhus in Greece and we highlight the importance of awareness of this difficult-to-recognize undifferentiated febrile illness.

Experience of a 40-day (6 week) LMWH treatment for isolated distal deep vein thrombosis.

Clinical significance of distal deep vein thrombosis (DVT) is important as it can potentially result in pulmonary embolism (PE), DVT extension, DVT recurrence and post-thrombotic syndrome (PTS). Controversy remains about the necessity and modalities of anticoagulation in all distal DVT. Evaluation of the efficiency of a 40-day weight-based low molecular weight heparin (LMWH) treatment in a cohort of 119 consecutive patients with distal DVT. Compression ultrasonography of the lower limb was performed initially for diagnosis as well as at the end of the treatment to identify persistence or resolution of the blood clot. A 3-month follow-up estimated the rates of PE, DVT recurrence, DVT extension, PTS and bleeding. Risk factors for DVT were considered to evaluate a possible correlation between them and the outcomes. In 71.4% of the patients the blood clot was totally dissolved and thrombus persistence was statistically associated with the number of initially involved veins. DVT recurrence occurred in 5% of patients and was also associated with the number of initial clotted veins. DVT extension and PTS rates were present in 1.7% and 3.4% respectively and no patient was diagnosed with PE or bleeding. This retrospective study including a limited number of patients and no control group supports that a 40-day weight-based LMWH treatment after distal DVT seems to be efficient when one single vein is initially affected whereas for multiple vein distal DVT and to avoid potential DVT recurrence, longer than 6 weeks of anticoagulant treatment is required. Our results support safety of the treatment, its potential to prevent DVT extension and the occurrence of PE.

Magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis.

This paper evaluated magnetic nanoparticle-enhanced PCR for the detection and identification of Staphylococcus aureus and Salmonella enteritidis. Two different types of magnetic nanoparticles designated MPIO (iron concentration 2.5 mg/ml, size 1 µm) and NP (iron concentration 8.7 mg/ml, size 60 nm), both conjugated with S. aureus or S. enteritidis antibodies were evaluated as an enrichment procedure for PCR-detection of the pathogens in Trypticase Soy Broth, milk, blood and meat broth. Bacterial suspensions (1.5x108 cfu/ml) were prepared and serial diluted 10-1. The MPIO and NP nanoparticles were added, followed by incubation for 1 hour at room temperature, magnetic separation of the pellet, DNA extraction and PCR, targeting the femA and invA sequences. The nanoparticle-free and the NP-supplemented dilutions were positive down to the 1.5x102 cfu/ml concentration for both bacteria. The MPIO-supplemented dilutions were positive down to approx. 2x100 cfu/ml concentration, respectively. Bacteria-free TSB was negative by PCR. MPIO nanoparticles (size 1 µm) enhanced the detection of S. aureus and S. enteritidis by PCR, whilst NP nanoparticles (size 60 nm) did not, thus indicating that the size of the magnetic nanoparticles play a significant role in the enrichment procedure.

Molecular detection of potential sexually transmitted pathogens in semen and urine specimens of infertile and fertile males.

A total of 93 infertile and 70 fertile men attending various urology and gynecology clinics in Jordan were investigated in this prospective study. First void urine and the corresponding semen specimens were collected from 96% of the patients. Presence of Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), and Mycoplasma genitalium (MG) DNA in specimen was detected using polymerase chain reaction. The distribution of NG, CT, UU, and MG in semen and FVU specimens among infertile versus fertile men was 6.5% versus 0%, 4.3% versus 1.4%, 10.8% versus 5.7%, and 3.2% versus 1.4%, respectively. Two of infertile and 1 of fertile men harbored mixed pathogens. The highest number of positive potential pathogens was found among young men aged 20-29 years old. The present study found a very high concordance between the detection of CT, UU, and MG DNA in semen and the corresponding FVU specimens, while NG DNA found only in semen and not in the corresponding FVU specimens. This study also revealed that Ureaplasma parvum species is more prevalent than Ureaplasma urealyticum in specimens of infertile men (90%). The study demonstrates that infertile men have higher prevalence of NG, CT, UU, and MG compared with fertile men and NG as significantly associated with infertile men.

The shotgun proteomic study of the human ThinPrep cervical smear using iTRAQ mass-tagging and 2D LC-FT-Orbitrap-MS: the detection of the human papillomavirus at the protein level.

The ThinPrep cervical smear is widely used in clinical practice for the cytological and molecular screening against abnormal cells and Human Papillomavirus (HPV) infection. Current advancements made to LC-MS proteomics include the use of stable isotope labeling for the in-depth analysis of proteins in complex clinical specimens. Such approaches have yet to be realized for ThinPrep clinical specimens. In this study, an LC-MS method based on isobaric (iTRAQ) labeling and high-resolution FT-Orbitrap mass spectrometry was used for the proteomic analysis of 23 human ThinPrep smear specimens. Tandem mass spectrometry analysis was performed with both nitrogen high collision dissociation (HCD MS/MS) and helium collision induced dissociation (CID MS/MS) peptide fragmentation modes. The analysis of three 8-plex sample sets yielded the identification of over 3200 unique proteins at FDR < 1%, of which over 2300 proteins were quantitatively profiled in at least one of the three experiments. The interindividual variability served to define the required sample size needed to identify significant protein expression differences. The degree of in-depth proteome coverage allowed the detection of 6 HPV-derived proteins including the high-risk HPV16 type in the specimens tested. The presence of the HPV strains of origin was also confirmed with PCR-hybridization molecular methods. This proof-of-principle study constitutes the first ever report on the nontargeted analysis of HPV proteins in human ThinPrep clinical specimens with high-resolution mass spectrometry. A further testament to the sensitivity and selectivity of the proposed study method was the confident detection of a significant number of phosphopeptides in these specimens.

Climate changes, environment and infection: facts, scenarios and growing awareness from the public health community within Europe.

Climate change is a current global concern and, despite continuing controversy about the extent and importance of causes and of its effects, it seems likely that it will affect the incidence and prevalence of both residual and imported infections in Europe. Climate affects mainly the range of infectious diseases, whereas weather affects the timing and intensity of outbreaks. Climate change scenarios include a change distribution of infectious diseases with warming and changes in outbreaks associated with weather extremes. The largest health impact from climate change for Europe doesn't come from vector borne infectious diseases. This does not mean that these types of health impacts will not arise in Europe. The ranges of several vector-borne diseases or their vectors are already changing in altitude due to warming. In addition, more intense weather events create conditions conductive to outbreaks of infectious diseases: Heavy rains leave insect breeding sites, drive rodents from burrows, and contaminate clean water systems. The incidence of mosquito-borne parasitic and viral diseases, are among those diseases most sensitive to climate. Climate change affect disease transmission by shifting the vector's geographic range and by shortening the pathogen incubation period. climate-related increases in temperature in sea surface and level would lead to higher incidence of waterborne infectious and toxin-related illnesses, such as cholera and seafood intoxication. Climate changes all around the world with impact in Europe are demonstrated by the fact that recent cases of cholera have been imported to Europe from Kenya, where spreading epidemic has been linked to the El Niño phenomenon, originated from the Pacific Ocean. Human migration and damage to health infrastructures from aberrant climate changes could indirectly contribute to disease transmission. Human susceptibility to infections might be further compounded by alterations in the human immune system caused by increased exposure to ultraviolet radiation and malnutrition due to alterations in agricultural products. Different kind of incidents in Europe with extreme weather events demonstrated effects on public health. The recent outbreak of the insect-borne Chikungunya virus in Italy in 2007 is an example of the kind of new health threat that the EU must be vigilant to confront. In addition, health effects of flooding, have been related to an excess cases of leptospirosis and campylobacter enteritis. Such examples have been demonstrated reported after flooding in the Czech Republic. Similarly, an increase of cryptosporidiosis in the United Kingdom has been related to flooding. Changing vector distributions associated with tickborne encephalitis and malaria have also been demonstrated in EU. A recently reported case of malaria in Italy in June 2008, suspected to be indigenously acquired, has shown how easily malaria could be reintroduced into several countries in the region. Another case of malaria in Greece in May 2010 affecting a young man living in a forestry region was claimed at KEELPNO-the Greek Center for disease control. Would this latest case be considered closely related to the one from Italy? If yes, then Public Health Services should elaborate plans to affront possible tickborne diseases. Heat waves are important causes of mortality on mortality are important. The deaths seen in France in 2003 from a heat wave are projected to be repeated, as heat waves become more severe. However, heat waves impacts on the transmission and severity of infectious diseases have not been elucidated. Finally scientific challenges include the elucudation of climate changes and extreme weather condition impact on infection transmission and outcome, human immune system changes and infection response, outbreak scenarios, animal and plant health and public health preparedness. European action plans to affront climate changes related health and infection problems are developed by the EU Commission at different levels and jointly by different DGs. In a few words within the EU the following points on human, animal and plant health are considered a priority: * Strengthening cooperation between the services of these three branches of health (human, animals, plants); * Developing action plans in the event of extreme weather conditions, in order to be better prepared and to react in the best way; * Gathering more reliable information on the risks of climate change whilst maintaining international cooperation, in particular with the WHO, as cooperation beyond that between Member States will be required to be more effective; * Providing additional effort to identify the most effective measures; * Improving the surveillance and the control of the animal diseases. The European Commission has decided to consider climate change, and the consequences it has on health, with greater importance whilst being aware that it is at the root of numerous diseases.

Comparison of cytology, colposcopy, HPV typing and biomarker analysis in cervical neoplasia.

BACKGROUND: Cervical cancer is the leading cause of mortality among women worldwide, despite existing prevention programs. In light of the recent development of anti-HPV vaccines, the aim of this study was to evaluate concurrently the efficacy of four methods for risk assessment (cytology, colposcopy, HPV molecular typing and detection of biomarkers in cervical biopsies) in an attempt to define the most efficient combination. PATIENTS AND METHODS: The studied group included 62 women with abnormal Pap tests and cervical lesions ranging from cervicitis and condylomas to intraepithelial neoplasias and invasive cancer. All women underwent full colposcopy assessment and colposcopically-taken biopsies were selected for histological examination, immunohistochemical identification of p16, p53, Bcl-2 biomarkers, as well as molecular detection and typing of HPV genomes. RESULTS: Cytology and colposcopy showed very high sensitivity in detecting CIN and cancer (91.7% and 94.4%, respectively), but low specificity (34.6% and 50%, respectively). The detection of the 3 biomarkers reached an impressive sensitivity (83.3%) and a moderate specificity (65.4%). HPV detection and typing achieved 77.8% sensitivity, and the highest specificity of 80.8% in detecting CIN and cancer cases. HPV DNA testing had the highest positive prognostic value (84.9%; confidence interval, CI: 67.4%- 94.3%) and cytology the lowest (66.0%; CI: 51.2%- 78.4%). Coupled HPV typing and colposcopy proved to be the most efficient combination, increasing sensitivity to 97.2% and negative prognostic value to 92.3%. The estimation of cervical neoplasia or cancer in women with high-risk HPV types increased approximately 15-fold (odds ratio, OR: 14.70; CI: 4.30-50.09, p<0.001), ~23-fold in the case of combined positive biomarkers (OR: 23.18; CI: 4.97- 104.23, p<0.001), and 35-fold in case of colposcopically detected cervical neoplasia (OR: 35.00; CI: 5.16- 225.07, p<0.001). CONCLUSION: The most efficient combination among all tested methodologies was found to be HPV typing with colposcopy.

Water-soluble amphotericin B-polyvinylpyrrolidone complexes with maintained antifungal activity against Candida spp. and Aspergillus spp. and reduced haemolytic and cytotoxic effects.

OBJECTIVES: Poor solubility and toxicity severely hinder the clinical use of amphotericin B (AmB), in spite of its attractive chemotherapeutic properties. Water-soluble complexes of AmB and polyvinylpyrrolidone (AmB-PVP) could display lower cytotoxicity while maintaining antifungal activity. METHODS: AmB-PVP [with PVP of 10, 24 and 40 kDa (AC1, AC2 and AC4)] were compared with free AmB for (i) activity against Candida spp. (five albicans; nine non-albicans) and Aspergillus spp. (four strains), (ii) haemolysis of sheep red blood cells, and (iii) release of lactate dehydrogenase from J774 macrophages [with further comparison with free PVP and a liposomal formulation of amphotericin (AmBisome)]. RESULTS: MICs and MFCs of AC1, AC2 and AC4 against Candida spp. and of AC2 and AC4 against Aspergillus spp. were similar to those of AmB (and even lower for some Candida strains). Killing kinetics (24 h) were also similar. Haemolytic activity of AC2 and AC4 was 2-fold lower than that of free AmB. Cytotoxicity of AC2 towards J774 macrophages was 8-fold lower, and that of AC4 5-fold lower than that of AmB and not significantly different from that of AmBisome. The lower cytotoxicity of AC2, AC4 was correlated with a lower cellular accumulation of amphotericin. Spectroscopic analysis shows that the lower toxicity of AmB-PVP was not owing to significant change in the monomeric/polymeric forms ratio of the drug. CONCLUSIONS: AmB-PVP complexes compared favourably with AmB for antifungal activity, were less haemolytic and cytotoxic than AmB, and show a similar cytotoxicity profile to AmBisome.

Could procalcitonin be a predictive biological marker in systemic fungal infections?. Study of 14 cases.

BACKGROUND: Procalcitonin (PCT) is a recently described inflammatory marker that has been shown to increase significantly in patients with severe systemic bacterial infections or sepsis. Reports on the diagnostic and predictive value of PCT in systemic fungal infections are limited. METHODS: In order to evaluate the role of PCT in systemic mycosis, 14 patients (mean age 40 years) with proven or probable systemic fungal infections were investigated. Blood samples were collected on days 1, 3, 5, and 10 after the onset of signs and symptoms of systemic fungal infection (clinical and/or laboratory diagnosis and/or radiographic evidence). PCT measurements were performed using an immunoluminometric assay. RESULTS: In five patients with severe fungal infection and an unfavorable course (patient group 2), PCT levels were moderately elevated on day 3 (0.5-1.0 ng/ml), whereas they were normal in the patients who recovered (patient group 1). High PCT levels (>/=1.11 ng/ml) were detected on the 10th day of the course of the illness in patient group 2. A normal or moderate elevation of PCT on day 10 was observed in patient group 1. The difference in mean PCT levels in patient groups 1 and 2 on days 3 and 10 were statistically significant. CONCLUSIONS: PCT levels seem to correlate with the severity and outcome of systemic fungal infection. If this finding can be confirmed in a larger number of patients, it could serve as a prognostic indicator.

New nystatin polymeric complexes and their in vitro antifungal evaluation in a model study with Fusarium oxysporum.

Six water-soluble nystatin-polyvilnylpyrrolidone complexes with respective MW of 10 kDa (NC1), 25 kDa (NC2), 30 kDa (NC3), 40 kda (NC4), 90 kDa (NC5), 360 kDa (NC6) were synthesized. The activity of the complexes was compared with that of nystatin against growth and spore germination of Fusarium oxysporum f.sp. radiciscucumerinum. The ED50 value (effective dose) of free nystatin in aqueous solution on growth inhibition on solid medium was determined at 35.7 ppm. The ED50 of the complexes NC3, NC4, NC5, and NC6 ranged from 2.2 to 4 times lower than that of nystatin. The NC6 complex exhibited the highest activity, followed by NC5, NC4, and NC3. The activities of NC1 and NC2 were about 3 and 1.7 times higher than nystatin respectively in the same in vitro model. The complexes NC6. NC1 and NC4 were 25.4, 13.6 and 6.9 times more active respectively than nystatin against spore germination of E oxysporum. The activity of the nystatin complexes was dependent on the molecular weight of the polymeric carrier.