Stalactites Core Prospect as Environmental "Microbial Ark": The Actinomycetota Diversity Paradigm, First Reported from a Greek Cave.

Speleothems found in caves worldwide are considered the natural libraries of paleontology. Bacteria found in these ecosystems are generally limited to Proteobacteria and Actinomycetota, but rare microbiome and "Dark Matter" is generally under-investigated and often neglected. This research article discusses, for the first time to our knowledge, the diachronic diversity of Actinomycetota entrapped inside a cave stalactite. The planet's environmental microbial community profile of different eras can be stored in these refugia (speleothems). These speleothems could be an environmental "Microbial Ark" storing rare microbiome and "Dark Matter" bacterial communities evermore.

Haematology reference values for Dicentrarchus labrax and Sparus aurata: A systematic review and meta-analysis.

Haematological parameters are frequently used as physiological indicators in aquaculture studies. These parameters also have extended applications in clinical evaluation, diagnosis and prognosis in fish health status. However, no normal reference range of values has been demonstrated in depth for any of these haematological parameters for the European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus aurata). The main objective of this article is to present for the first time through extended literature review, the haematological parameters normal range values for the two most important aquaculture fish species farmed in Mediterranean Sea, D. labrax and S. aurata, and to demonstrate their similarities and their differences. In this article, we also discuss the environmental and external factors affecting their normal blood parameters values and we propose fundamental guidelines on the reporting units.

Metataxonomic Analysis of Bacteria Entrapped in a Stalactite's Core and Their Possible Environmental Origins.

Much is known about microbes originally identified in caves, but little is known about the entrapment of microbes (bacteria) in stalactites and their possible environmental origins. This study presents data regarding the significant environmental distribution of prokaryotic bacterial taxa of a Greek stalactite core. We investigated the involvement of those bacteria communities in stalactites using a metataxonomic analysis approach of partial 16S rRNA genes. The metataxonomic analysis of stalactite core material revealed an exceptionally broad ecological spectrum of bacteria classified as members of Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and other unclassified bacteria. We concluded that (i) the bacterial transport process is possible through water movement from the upper ground cave environment, forming cave speleothems such as stalactites, (ii) bacterial genera such as Polaromonas, Thioprofundum, and phylum Verrucomicrobia trapped inside the stalactite support the paleoecology, paleomicrobiology, and paleoclimate variations, (iii) the entrapment of certain bacteria taxa associated with water, soil, animals, and plants such as Micrococcales, Propionibacteriales, Acidimicrobiales, Pseudonocardiales, and α-, ß-, and γ-Proteobacteria.

Urine HPV in the Context of Genital and Cervical Cancer Screening-An Update of Current Literature.

Within the previous decades, following the widespread implementation of HPV-related biomarkers and computerization in liquid-based cytology, screening for lower genital tract malignancies has been optimized in several parts of the world. Many organized anogenital cancer prevention systems have reached a point at which efficacy is more a matter of population coverage and less of available infrastructures. Meanwhile, self-sampling modalities in which biologic material (vaginal secretions, urine, etc.) is obtained by the individual and not the clinician and subsequently undergoes examination for HPV biomarkers enjoy appreciating acceptance. Bygone the initial skepticism that vaginal or urine HPV represents "passenger" transient infections, extensive scientific work has been conducted to optimize high-risk HPV (hrHPV) detection from this "novel" biologic material. Nowadays, several state-of-the-art meta-analyses have illustrated that self-sampling techniques involving urine self-sampling represent a feasible alternative strategy with potentially enhanced population coverage possessing excellent performance and sensitivity. Recently published scientific work focusing on urine HPV was reviewed, and after a critical appraisal, the following points should be considered in the clinical application of hrHPV urine measurements; (i) use of first-void urine (FVU) and purpose-designed collection devices; (ii) using a preservation medium to avoid human/HPV DNA degradation during extraction and storage; (iii) using polymerase chain reaction (PCR) based assays, ideally with genotyping capabilities; (iv) processing of a sufficient volume of whole urine; and (v) the use of an analytically sensitive HPV test/recovery of cell-free HPV DNA in addition to cell-associated DNA.

Acceptability of Self-Sampling for Human Papillomavirus-Based Cervical Cancer Screening.

Background: Human papillomavirus (HPV)-DNA testing combined with self-sampling could increase cervical cancer screening effectiveness, utilizing a sensitive screening modality and an easy sampling method with minimal pain or discomfort. Self-sampling acceptability, though, is pivotal. Materials and Methods: This study is a nested cross-sectional survey within GRECOSELF, a cross-sectional study on HPV-based screening with self-sampling, aiming at investigating self-sampling acceptability among Greek women residing in rural areas, and the factors affecting it. Women between 25 and 60 years old were recruited by midwives participating in a nationwide midwifery network. Participants, after self-sampling, filled out a questionnaire with three sections, one regarding demographic characteristics, a second with questions pertaining to the participants' cervical cancer screening history, and a third with questions regarding the self-sampling process per se. Results: The sample included 13,111 women. Most participants (67.9%), including those screened or not in the past, would prefer self-sampling if assured that the results are not inferior to standard testing. Discomfort or pain during self-sampling was absent or minimal in 97.1% and 96.5% of the cases, respectively, and 74.4% of the women felt adequately confident that they followed the instructions correctly. Women mostly preferred self-sampling at home compared with health care facilities. Pain and discomfort during the procedure, although rare, were significant factors against acceptance. Most of the women reporting a negative impression had a negative experience with conventional sampling in the past. Conclusion: Self-sampling is highly acceptable. Acceptance can be further improved with proper communication of the process and its noninferiority compared with conventional screening.

Alterations of HPV-Related Biomarkers after Prophylactic HPV Vaccination. A Prospective Pilot Observational Study in Greek Women.

The objective of this study was to investigate the hypothesis that HPV vaccination administered in patients with low-grade (LG) cytology shortly after an initial colposcopic assessment could prospectively alter HPV-related biomarkers. This was a prospective pilot observational study involving women attending a colposcopy clinic for evaluation of abnormal LG cytology that were advised to undergo HPV vaccination and proceeded accordingly. These women were compared with a matched unvaccinated group. Women requiring cervical biopsies or CIN treatment were excluded. INTERVENTION: A full three-dose HPV vaccination was undertaken with either the 2-valent or the 4-valent anti-HPV VLP vaccine. LBC samples were obtained prior and after the completion of the vaccination regimen and tested for HPV DNA genotyping (CLART-2 HPV test) and E6 and E7 mRNA (NASBA technique). RESULTS: Alterations of HPV-related biomarkers at a colposcopy reassessment appointment 12 months later. ANALYSIS: The p-values, relative risk (RR), absolute relative risk (ARR), number needed to treat (NNT) and 95% confidence intervals for each biomarker in each group were assessed. RESULTS: A total of 309 women were included in the analysis. One hundred fifty-two women received the vaccine. HPV vaccination reduced in a statistically significant manner (p < 0.05) HPV DNA positivity rates for genotypes 16, 18, and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18, and 31 genotypes, respectively, prior to vaccination. A less pronounced, statistically insignificant reduction was shown for women who tested positive for both HPV DNA and mRNA E6 and E7 expression for HPV16, 18, and 33 subtypes. Statistically significant reduction in HPV mRNA positivity was solely documented for genotype 31 (p = 0.0411). CONCLUSIONS: HPV vaccination appears to significantly affect the rates of HPV16, 18, and 31 DNA-positive infections in the population testing HPV DNA-positive for the aforementioned genotypes. The above findings deserve verification in larger cohorts.

Assessment of HPV Risk Type in H&E-stained Biopsy Specimens of the Cervix by Microscopy Image Analysis.

OBJECTIVES: The objective of this study was (a) to identify, by computer processing of digitized images of hematoxylin and eosin (H&E)-stained biopsy material of the cervix, differences in the structure of nuclei between high-risk (HR) and low-risk (LR) human papillomavirus virus (HPV) types and (b) to assess the HPV risk type by designing a decision-support system (DSS). MATERIALS AND METHODS: Clinical material comprised H&E-stained biopsies from squamous intraepithelial lesions of 55 patients with polymerase chain reaction-verified HR-HPV (26 patients) or LR-HPV (29 patients) infection. From each patient's biopsy specimen, we digitized 1 region of interest, guided by the expert physician. After the segmentation of nuclei, we quantified from each nucleus 77 textural and morphologic features. We represented each patient by a 77-feature vector, the feature means of all nuclei, and we created 2 classes for HR-HPV and LR-HPV types. We carried out (a) a statistical analysis to determine features with statistically significant differences between the 2 classes and (b) a discriminant analysis, by designing a DSS, to estimate the HPV risk type. RESULTS: Statistical analysis revealed 40 features with between-classes statistically significant differences and discriminant analysis showed that the best DSS design achieved a high accuracy of about 93% in identifying the HPV risk type on data not used in the design of the DSS. CONCLUSIONS: Nuclei of HR-HPV types were of higher intensity, contained larger structures, had higher edges, were coarser, rougher, had higher contrast, were larger, and attained more irregular shapes. The proposed DSS indicates that discrimination of HPV risk type from images of H&E-stained biopsy material of the cervix is promising.

GPU-enabled design of an adaptable pattern recognition system for discriminating squamous intraepithelial lesions of the cervix.

The aim of the present study was to design an adaptable pattern recognition (PR) system to discriminate low- from high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively) of the cervix using microscopy images of hematoxylin and eosin (H&E)-stained biopsy material from two different medical centers. Clinical material comprised H&E-stained biopsies of 66 patients diagnosed with LSIL (34 cases) or HSIL (32 cases). Regions of interest were selected from each patient's digitized microscopy images. Seventy-seven features were generated, regarding the texture, morphology and spatial distribution of nuclei. The probabilistic neural network (PNN) classifier, the exhaustive search feature selection method, the leave-one-out (LOO) and the bootstrap validation methods were used to design the PR system and to assess its precision. Optimal PR system design and evaluation were made feasible by the employment of graphics processing unit (GPU) and Compute Unified Device Architecture (CUDA) technologies. The accuracy of the PR-system was 93% and 88.6% when using the LOO and bootstrap validation methods, respectively. The proposed PR system for discriminating LSIL from HSIL of the cervix was designed to operate in a clinical environment, having the capability of being redesigned when new verified cases are added to its repository and when data from other medical centers are included, following similar biopsy material preparation procedures.

Implementation of HPV-based Cervical Cancer Screening Combined with Self-sampling Using a Midwifery Network Across Rural Greece: The GRECOSELF Study.

Self-sampling for human papillomavirus (HPV) testing is an alternative to physician sampling particularly for cervical cancer screening nonattenders. The GRECOSELF study is a nationwide observational cross-sectional study aiming to suggest a way to implement HPV-DNA testing in conjunction with self-sampling for cervical cancer screening in Greece, utilizing a midwifery network. Women residing in remote areas of Greece were approached by midwives, of a nationwide network, and were provided with a self-collection kit (dry swab) for cervicovaginal sampling and asked to answer a questionnaire about their cervical cancer screening history. Each sample was tested for high-risk (hr) HPV with the Cobas HPV test. HrHPV-Positive women were referred to undergo colposcopy and, if needed, treatment according to colposcopy/biopsy results. Between May 2016 and November 2018, 13,111 women were recruited. Of these, 12,787 women gave valid answers in the study questionnaire and had valid HPV-DNA results; hrHPV prevalence was 8.3%; high-grade cervical/vaginal disease or cancer prevalence was 0.6%. HrHPV positivity rate decreased with age from 20.7% for women aged 25-29 years to 5.1% for women aged 50-60 years. Positive predictive value for hrHPV testing and for HPV16/18 genotyping ranged from 5.0% to 11.6% and from 11.8% to 27.0%, respectively, in different age groups. Compliance to colposcopy referral rate ranged from 68.6% (for women 25-29) to 76.3% (for women 40-49). For women residing in remote areas of Greece, the detection of hrHPV DNA with the Cobas HPV test, on self-collected cervicovaginal samples using dry cotton swabs, which are provided by visiting midwives, is a promising method for cervical cancer secondary prevention.

Multifeature Quantification of Nuclear Properties from Images of H&E-Stained Biopsy Material for Investigating Changes in Nuclear Structure with Advancing CIN Grade.

Background: Cervical dysplasia is a precancerous condition, and if left untreated, it may lead to cervical cancer, which is the second most common cancer in women. The purpose of this study was to investigate differences in nuclear properties of the H&E-stained biopsy material between low CIN and high CIN cases and associate those properties with the CIN grade. Methods: The clinical material comprised hematoxylin and eosin- (H&E-) stained biopsy specimens from lesions of 44 patients diagnosed with cervical intraepithelial neoplasia (CIN). Four or five nonoverlapping microscopy images were digitized from each patient's H&E specimens, from regions indicated by the expert physician. Sixty-three textural and morphological nuclear features were generated for each patient's images. The Wilcoxon statistical test and the point biserial correlation were used to estimate each feature's discriminatory power between low CIN and high CIN cases and its correlation with the advancing CIN grade, respectively. Results: Statistical analysis showed 19 features that quantify nuclear shape, size, and texture and sustain statistically significant differences between low CIN and high CIN cases. These findings revealed that nuclei in high CIN cases, as compared to nuclei in low CIN cases, have more irregular shape, are larger in size, are coarser in texture, contain higher edges, have higher local contrast, are more inhomogeneous, and comprise structures of different intensities. Conclusion: A systematic statistical analysis of nucleus features, quantified from the H&E-stained biopsy material, showed that there are significant differences in the shape, size, and texture of nuclei between low CIN and high CIN cases.

Activity of Tigecycline in combination with Colistin, Meropenem, Rifampin, or Gentamicin against KPC-producing Enterobacteriaceae in a murine thigh infection model.

Limited antimicrobials remain active for treating severe infections due to KPC-producing pathogens, and optimal regimens have not been established. In murine thigh infections caused by nine KPC-producing clinical strains of Enterobacteriaceae (meropenem MICs, 1 to 4 µg/ml), we evaluated the activities of tigecycline, colistin, meropenem, rifampin, and gentamicin in single and combination regimens lasting for 24 h and 48 h. Rifampin, tigecycline, and gentamicin were the most effective monotherapies, reducing significantly the CFU counts yielded from thighs infected by 88.9 to 100%, 77.8 to 88.9%, and 66.7 to 88.9% of strains, respectively; meropenem and colistin alone exhibited considerably lower performance (significant CFU reduction in 33.3% and 22.2 to 33.3% of the strains, respectively). The addition of rifampin or gentamicin to tigecycline produced synergistic effect in most strains, while antagonism was observed in 33.3 to 44.4% of the strains when colistin was added to tigecycline and in 44.4 to 55.5% of the strains for meropenem combination with tigecycline. Tigecycline combinations with gentamicin or with rifampin caused higher CFU reductions than did tigecycline plus colistin or plus meropenem with almost all strains. Furthermore, tigecycline plus gentamicin was significantly more effective than tigecycline plus colistin or tigecycline plus meropenem in 33.3 to 44.4% and 55.5 to 66.7% of the strains, respectively, while tigecycline plus rifampin significantly outperformed tigecycline plus colistin and tigecycline plus meropenem in 33.3% and 66.7 to 77.8% of the strains, respectively. Overall, our in vivo study showed that tigecycline plus rifampin or plus gentamicin is a robust regimen against soft tissue infections caused by KPC-producing strains. The combinations of tigecycline with colistin or meropenem should be considered with caution in clinical practice.

Virulence of Acinetobacter baumannii Exhibiting Phenotypic Heterogeneous Growth against Meropenem in a Murine Thigh Infection Model.

Acinetobacter baumannii may exhibit phenotypic heterogeneous growth under exposure to antibiotics. We investigated the in vitro characteristics of A. baumannii isolates grown heterogeneously in the presence of meropenem and their virulence evaluated in experimental infections treated with meropenem. Five clinical A. baumannii isolates and the respective heterogeneously grown subpopulations were tested by agar dilution minimum inhibitory concentration (MIC) testing, pulsed field gel electrophoresis (PFGE), population analysis using meropenem and growth curves. The virulence of isolates and the therapeutic efficacy of three meropenem dosing schemes was evaluated in a neutropenic murine thigh infection model. The clinical isolates were meropenem-susceptible (MICs 1 to 4 mg/liter) and exhibited three distinct PFGE patterns. In all clinical isolates, population analysis yielded heterogeneously grown colonies. After seven subcultures in antibiotic-free media, resistant MIC levels were retained in two isolates (heteroresistant), while three isolates were reversed to susceptible MICs (persisters). Clinical isolates and heterogeneous subpopulations had similar growth rates. The heterogeneously grown A. baumannii subpopulations had reduced virulence, killing considerably fewer animals than the respective clinical isolates without treatment. The meropenem treatment outcome was similar in infections caused by the clinical and the heterogeneous isolates, irrespective to their MICs. In vitro meropenem exposure induces phenotypic heterogeneous growth in A. baumannii. Compared with the parental clinical isolates, the heterogeneously grown subpopulations exhibited lower virulence, killing fewer mice and responding equally to meropenem treatment, despite their higher MICs.

Activity of oxacillin versus that of vancomycin against oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates evaluated by population analyses, time-kill assays, and a murine thigh infection model.

We compared the activity of dicloxacillin with that of vancomycin against 15 oxacillin-susceptible, methicillin-resistant Staphylococcus aureus (OS-MRSA) clinical isolates. By population analyses, we found that 6 OS-MRSA isolates were able to grow in the presence of up to 8 µg/ml dicloxacillin and 9 isolates were able to grow in 12 to >32 µg/ml dicloxacillin; all isolates grew in up to 2 µg/ml vancomycin. Both drugs exhibited similar bactericidal activities. In experimental infections, the therapeutic efficacy of dicloxacillin was significant (P < 0.05 versus untreated controls) in 10 OS-MRSA isolates and vancomycin was effective (P < 0.05) against 12 isolates; dicloxacillin had an efficacy that was comparable to that of vancomycin (P > 0.05) in 8 isolates. The favorable response to dicloxacillin treatment might suggest that antistaphylococcal penicillins could be used against OS-MRSA infections.

In vitro and in vivo evaluations of oxacillin efficiency against mecA-positive oxacillin-susceptible Staphylococcus aureus.

Community-type Staphylococcus aureus strains that are positive for mecA and PBP2a but appear phenotypically susceptible to oxacillin are increasingly reported worldwide. Four S. aureus clinical isolates carrying the mecA gene with oxacillin MICs of <2 microg/ml were tested for oxacillin efficiency by population analyses and experimental thigh infections. These isolates harbored staphylococcal cassette chromosome mec type IV and belonged to two genotypes. Two of the four isolates were found by population analysis to be truly oxacillin susceptible. All four isolates exhibited significant reductions in the numbers of colonies grown after dicloxacillin treatment of experimental thigh infections, as also did a mecA-negative S. aureus control strain. These observations indicate that some of the phenotypically oxacillin susceptible mecA-positive Staphylococcus aureus isolates may be at least partially responsive to oxacillin.