Mutational Profiles of Cutaneous Squamous Cell Carcinomas with Different Patterns of Clinical Aggression from Head and Neck Regions.

Cutaneous squamous cell carcinoma is a prevalent malignancy with a rising incidence and a notably high mutational load. Exploring the genetic nuances of cSCC and investigating molecular approaches stands as a potential avenue for improving outcomes in high-risk patients. This retrospective case-control study involved two cohorts, one of 14 patients (the "discovery cohort") and the other of 12 patients (the "validation cohort"), with cSCC located in the head/neck anatomical region and diagnosed at the pT2 stage. Overall, cases developed early local relapses of the disease, whereas controls never relapsed during the entire follow-up period. A next-generation sequencing (NGS) approach conducted on histological samples revealed that TP53 and CDKN2A were the most frequently mutated genes in our series. No specific mutations were identified as potential prognostic or therapeutic targets. Controls exhibited a tendency toward a higher mutational rate compared to cases. It is possible that an increased number of mutations could prompt the cSCC to expose more antigens, becoming more immunogenic and facilitating recognition by the immune system. This could enhance and sustain the immunological response, potentially preventing future recurrences.

Multidisciplinary Management of Cutaneous Squamous Cell Carcinoma of the Scalp: An Algorithm for Reconstruction and Treatment.

Background: Scalp-associated cutaneous squamous cell carcinoma (cSCC) presents formidable treatment challenges, especially when it leads to full-thickness defects involving bone. Aggressive or recurring cases often demand a multidisciplinary approach. Leveraging our surgical experience and a literature review, we introduce a therapeutic algorithm to guide the selection of reconstruction methods, particularly for locally advanced lesions, furthermore showing the synergy between surgery and other therapies for comprehensive, multidisciplinary disease management. Methods: Our algorithm stems from a retrospective analysis of 202 patients undergoing scalp cSCC resection and reconstruction over a 7-year period, encompassing 243 malignancies. After rigorous risk assessment and documentation of surgical procedures, reconstruction methods were therefore related to malignancy extent, depth, and individual clinical status. Results: The documented reconstructions included 76 primary closures, 115 skin grafts, 7 dermal substitute reconstructions, 33 local flaps, 1 locoregional flap, and 1 microsurgical free flap. Patients unsuitable for surgery received radiotherapy or immunotherapy after histological confirmation. Precise analysis of tumor characteristics in terms of infiltration extent and depth guided the selection of appropriate reconstruction and treatment strategies Combining these insights with an extensive literature review enabled us to formulate our algorithm for managing scalp cSCCs. Conclusions: Effectively addressing scalp cSCC, especially in locally advanced or recurrent cases, demands a systematic approach integrating surgery, radiotherapy, and immunotherapy. Our multidisciplinary team's decision-making algorithm improved patient outcomes by offering a broader spectrum of therapeutic options that can synergistically achieve optimal results.

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

Push-out bond strength of an experimental self-adhesive resin cement.

The adhesion of fiber posts luted with simplified adhesive systems has been a matter of great interest over the past years. The aim of this study was to assess the post retentive potential of experimental self-adhesive resin cement (EXP) when used alone and in combination with a self-etch adhesive. Fiber posts were placed in endodontically treated teeth and divided into four groups (n = 6) according the luting material, as follows: group 1 (EXP alone); group 2 (EXP used with self-etch adhesive); group 3 (marketed dual-cured cement used with self-etch adhesive); and group 4 (marketed self-adhesive cement). The push-out test was used to assess the retentive strength of fiber posts (expressed in MPa), and specimens were analyzed under a stereomicroscope to determine failure mode. The adhesive interface between the cement and root canal dentin for each group was evaluated using scanning electron microscopy. The post retentive potential of group 1 (EXP) (7.48 ± 4.35 MPa) was comparable with that of marketed cements from group 4 (6.79 ± 3.68 MPa) and group 3 (8.77 ± 4.58 MPa). When EXP was used in combination with self-etch adhesive (group 2), significantly higher push-out bond-strength values were measured (15.87 ± 4.68 MPa) compared with the other groups.

Squamous cell carcinoma of the scalp: a combination of different therapeutic strategies.

A case study of a 71-year-old man with a giant cutaneous squamous cell carcinoma of the scalp and calvaria is presented, where a combination of surgical excision, reconstruction with a latissimus dorsi muscular free flap, immunotherapy, and radiotherapy were used to control the disease for two years without recurrence.

Multidrug-resistant Proteus mirabilis bloodstream infections: risk factors and outcomes.

Our aims were to identify (i) risk factors associated with the acquisition of multidrug-resistant (MDR, to 3 or more classes of antimicrobials) Proteus mirabilis isolates responsible for bloodstream infections (BSIs) and (ii) the impact on mortality of such infections. Risk factors for acquiring MDR P. mirabilis BSIs were investigated in a case-case-control study; those associated with mortality were assessed by comparing survivors and nonsurvivors in a cohort study. The population consisted of 99 adult inpatients with P. mirabilis BSIs identified by our laboratory over an 11-year period (1999 to 2009), 36 (33.3%) of which were caused by MDR strains, and the overall 21-day mortality rate was 30.3%. Acquisition of an MDR strain was independently associated with admission from a long-term care facility (odds ratio [OR], 9.78; 95% confidence interval [CI], 1.94 to 49.16), previous therapy with fluoroquinolones (OR, 5.52; 95% CI, 1.30 to 23.43) or oxyimino-cephalosporins (OR, 4.72; 95% CI, 1.31 to 16.99), urinary catheterization (OR, 3.89; 95% CI, 1.50 to 10.09), and previous hospitalization (OR, 2.68; 95% CI, 10.4 to 6.89). Patients with MDR P. mirabilis BSIs received inadequate initial antimicrobial therapy (IIAT, i.e., treatment with drugs to which the isolate displayed in vitro resistance) more frequently than those with non-MDR infections; they also had increased mortality and (for survivors) longer post-BSI-onset hospital stays. In multivariate regression analysis, 21-day mortality was associated with septic shock at BSI onset (OR, 12.97; 95% CI, 32.2 to 52.23), P. mirabilis isolates that were MDR (OR, 6.62; 95% CI, 16.4 to 26.68), and IIAT (OR, 9.85; 95% CI, 26.7 to 36.25), the only modifiable risk factor of the 3. These findings can potentially improve clinicians' ability to identify P. mirabilis BSIs likely to be MDR, thereby reducing the risk of IIAT--a major risk factor for mortality in these cases--and facilitating the prompt implementation of appropriate infection control measures.

Direct maldi-tof mass spectrometry assay of blood culture broths for rapid identification of Candida species causing bloodstream infections: an observational study in two large microbiology laboratories.

We evaluated the reliability of the Bruker Daltonik's MALDI Biotyper system in species-level identification of yeasts directly from blood culture bottles. Identification results were concordant with those of the conventional culture-based method for 95.9% of Candida albicans (187/195) and 86.5% of non-albicans Candida species (128/148). Results were available in 30 min (median), suggesting that this approach is a reliable, time-saving tool for routine identification of Candida species causing bloodstream infection.

Performance of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group.

OBJECTIVE: The DIVA study is aimed at setting up a standardized genotypic tropism-testing on proviral-DNA for the routine clinical diagnostic-laboratory. METHODS: Twelve local centres and 5 reference centres (previously cross-validated) were identified. For inter-center validation-procedure, 60 peripheral-blood mononuclear cells (PBMCs) aliquots from 45 HAART-treated patients were randomly chosen for population V3 sequencing on proviral-DNA at local HIV centre and at reference-laboratory. Viral tropism was predicted by Geno2Pheno algorithm (False Positive Rate [FPR] = 20%) as proposed by the European-Guidelines. Quantification of total HIV-1 DNA was based on a method described by Viard (2004). RESULTS: Quantification of HIV-1 DNA was available for 35/45 (77.8%) samples, and gave a median value of 598 (IQR:252- 1,203) copies/10 PBMCs. A total of 56/60 (93.3%) samples were successfully amplified by both the reference and the local virological centers. The overall concordance of tropism prediction between local and reference centers was 54/56 (96.4%). Results of tropism prediction by local centers were: 33/54 (61.1%) R5 and 21/54 (38.9%) X4/DM. CONCLUSION: There was high concordance in the genotypic tropism prediction based on proviral DNA among different virological centers throughout Italy. Our results are in line with other European studies, and support the use of genotypic tropism testing on proviral DNA in patients with suppressed plasma HIV-1 RNA candidate to CCR5-antagonist treatment.

Tuberculosis in sheltered homeless population of Rome: an integrated model of recruitment for risk management.

The authors show the results of an integrated model for risk management of tuberculosis in a sample of sheltered homeless in Rome. Tuberculin skin test (TST) was used for evaluating the prevalence of latent infection (LTBI). In TST positives, expectorate was collected and chest X-ray was achieved. Multiple logistic regression analysis was performed to investigate determinants of infection. Out of 288 recruited subjects, 259 returned for the TST reading; 45.56% were positive and referred to a specialized center; 70 accessed the health facility and completed the clinical pathway. The risk factors associated to LTBI were male gender (OR = 3.72), age over 60 years (OR = 3.59), immigrant status (OR = 3.73), and obesity (OR = 2.19). This approach, based on an integrated social network, guarantees high adherence to screening (89.93%), allowing patients testing positive for latent tuberculosis infection to be diagnosed and rapidly referred to a specialized center.

Liquid Biopsy in the Oncological Management of a Histologically Undiagnosed Lung Carcinoma: A Case Report.

Lung cancer is one of the most common and lethal cancers worldwide. Numerous medications targeting specific molecular alterations in non-small cell lung cancer have been introduced in the last decade and have revolutionized the clinical management of the disease. Their use has brought to a parallel evolution of molecular testing techniques to identify alterations in druggable molecular targets within the genetic material of the tumors. To perform molecular testing, biopsy or surgery tissue specimens are needed, which in addition allow the histological characterization of the tumors. Unfortunately, in real-life practice not all the patients are suitable for biopsy or surgery procedures. The use of liquid biopsy for blood extracted tumoral DNA analysis is a promising approach in unbiopsied cases, but it is also weighted by several methodological and technical limitations. We report here a case of histologically undiagnosed lung cancer managed with a liquid biopsy and subsequently with anti-EGFR treatment. Our report highlights that the use of liquid biopsy molecular testing in specific clinical situations can offer treatment opportunities for fragile patients affected by lung cancer.

In vitro activities of anidulafungin and other antifungal agents against biofilms formed by clinical isolates of different Candida and Aspergillus species.

We tested the activities of anidulafungin and other antifungal agents against clinical isolates of different fungal species. For Candida species, high sessile MIC90s (SMIC90s) were obtained for fluconazole, voriconazole, and amphotericin B, whereas the anidulafungin SMIC90s were very low, as were those for caspofungin. Comparatively, for Aspergillus species, higher SMIC90 values were obtained not only for amphotericin B and voriconazole but also for the echinocandins.

Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia.

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severe nosocomial and community-acquired infections. Although enterococcal meningitis is rare, mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of this infection remain poorly understood, even though the ability of E. faecalis to avoid or survive phagocytic attack in vivo may be very important during the infection process. We previously showed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis was implicated in oxidative stress responses and, interestingly, in the survival within mouse peritoneal macrophages using an in vivo-in vitro infection model. In the present study, we investigated the role of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. By using an in vitro infection model, murine microglial cells were challenged in parallel with the wild-type strain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosed by microglia efficiently and to a similar extent, the ΔsodA mutant was found to be significantly more susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protection assay. In addition, a significantly higher percentage of acidic ΔsodA-containing phagosomes was found and these also underwent enhanced maturation as determined by the expression of endolysosomal markers. In conclusion, these results show that the MnSOD of E. faecalis contributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity, and this could even be important for intracellular killing in neutrophils and thus for E. faecalis pathogenesis.

Uncommon Neosartorya udagawae fungus as a causative agent of severe corneal infection.

We report the first documented case of a posttraumatic fungal keratitis caused by Neosartorya udagawae. The patient was empirically treated with fluconazole until a corneal scraping grew an Aspergillus fumigatus-like fungus, and itraconazole therapy was then established. A sequence-based approach assigned the isolate to the species. Five months after completion of antifungal therapy, endophthalmitis occurred and orbital exenteration was necessary.

Diagnosis of invasive aspergillosis by a commercial real-time PCR assay for Aspergillus DNA in bronchoalveolar lavage fluid samples from high-risk patients compared to a galactomannan enzyme immunoassay.

Culture-independent molecular techniques such as real-time PCRs offer the potential for early diagnosis of invasive aspergillosis (IA), thereby reducing the disease-associated mortality rate. PCR-based testing is presently excluded from disease-defining consensus criteria due to lack of standardization and clinical validation. A single-center prospective study was conducted to investigate the performance of the commercially available MycAssay Aspergillus test for detecting Aspergillus DNA in patients with suspicion of IA. To this end, a total of 158 bronchoalveolar lavage (BAL) fluid specimens that were consecutively collected from hematology (n = 68) and intensive care unit (n = 90) patients were examined. Sixteen of 17 (94.1%) specimens from patients with proven/probable IA were MycAssay positive, and 15 of these 16 patients were also positive by an "in-house" PCR assay. A total of 139 of 141 (98.6%) specimens from patients without proven/probable IA were MycAssay negative. Fifteen of 16 (94.1%) MycAssay-positive patients were also positive for BAL fluid galactomannan (GM) at an index cutoff of ≥1.0 (index range, 1.1 to 8.3), as were 3 patients without IA but with pulmonary fusariosis. Interestingly, in seven of the PCR-positive BAL specimens that tested culture positive for Aspergillus species, cycle threshold values were earlier than those of specimens with a culture-negative result. In conclusion, the MycAssay Aspergillus PCR appears to be a sensitive and specific molecular test for the diagnosis of IA, and its performance is comparable to that of the GM assay. However, more large studies are necessary to firmly establish its clinical utility in high-risk settings.

Gain of function mutations in CgPDR1 of Candida glabrata not only mediate antifungal resistance but also enhance virulence.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

Lysophosphatidic acid enhances antimycobacterial response during in vivo primary Mycobacterium tuberculosis infection.

Lysophospholipids may play an important protective role during primary infection of Mycobacterium tuberculosis (MTB) by enhancing innate antimycobacterial immune response of both macrophages and alveolar epithelial cells. Here, we show that treatment with lysophosphatidic acid (LPA) of mice aerogenically infected with MTB immediately after infection results in a significant early reduction of pulmonary CFUs and of histopathological damage in comparison with control mice. In contrast, treatment of acute disease does not result in any improvement of both microbiological and histopathological parameters. Altogether, these results show that LPA treatment can exert protective effect if administrated during primary infection, only.

Analysis of heat-induced changes in protein expression of Stenotrophomonas maltophilia K279a reveals a role for GroEL in the host-temperature adaptation.

Stenotrophomonas maltophilia is a microorganism of environmental and clinical importance as well as a frequent airway colonizer of cystic fibrosis (CF) individuals. We combined 2-DE and MALDI-TOF MS to profile the protein expression in S. maltophilia K279a, a completely sequenced clinical isolate, grown at 37 °C with respect to the strain grown at 26 °C. Among the proteins up-regulated at 37 °C, we identified GroEL, a molecular chaperone that mainly assist the folding and unfolding of proteins under both normal and stress conditions. A 2.4-kb groESL mRNA was detected independently by Northern blot analyses with a groES- and a groEL-specific probe, indicating that S. maltophilia groES and groEL form an operon. Primer extension analysis of S. maltophilia groESL done in Escherichia coli showed that 2 promoters, Pσ(32) and Pσ(70), were utilized under the heat-shock and normal condition, respectively, whereas S. maltophilia groEL was shown to act as a heat-shock gene at 37 °C, 42 °C, and, to a lesser extent, at 50 °C by real-time RT-PCR analyses. Finally, immunoblot analyses revealed that S. maltophilia GroEL strongly reacted with sera from CF patients chronically infected by the microorganism, but did not with sera from CF patients with sporadic infection or uninfected.

Genome-wide expression profiling of the response to short-term exposure to fluconazole in Cryptococcus neoformans serotype A.

BACKGROUND: Fluconazole (FLC), a triazole antifungal drug, is widely used for the maintenance therapy of cryptococcal meningoencephalitis, the most common opportunistic infection in AIDS patients. In this study, we examined changes in the gene expression profile of the C. neoformans reference strain H99 (serotype A) following FLC treatment in order to investigate the adaptive cellular responses to drug stress. RESULTS: Simultaneous analysis of over 6823 transcripts revealed that 476 genes were responsive to FLC. As expected up-regulation of genes involved in ergosterol biosynthesis was observed, including the azole target gene ERG11 and ERG13, ERG1, ERG7, ERG25, ERG2, ERG3 and ERG5. In addition, SRE1 which is a gene encoding a well-known regulator of sterol homeostasis in C. neoformans was up-regulated. Several other genes such as those involved in a variety of important cellular processes (i.e. lipid and fatty acid metabolism, cell wall maintenance, stress and virulence) were found to be up-regulated in response to FLC treatment. Conversely, expression of AFR1, the major transporter of azoles in C. neoformans, was not regulated by FLC. CONCLUSIONS: Short-term exposure of C. neoformans to FLC resulted in a complex altered gene expression profile. Some of the observed changes could represent specific adaptive responses to the antifungal agent in this pathogenic yeast.

Prospective evaluation of epidemiological, clinical, and microbiological features of pandemic influenza A (H1N1) virus infection in Italy.

Since several characteristics of pandemic influenza A (H1N1) virus infection remain to be determined, this study aimed to describe clinical features and complications of patients infected with H1N1. Subjects affected by influenza-like illnesses and a control group of asymptomatic patients were enrolled prospectively at an Emergency Department from October 2009 to April 2010. At enrollment, clinical data and nasopharyngeal swabs for virological analyses were obtained. Ill subjects were followed until recovery and swabs were collected weekly in patients infected with H1N1. Of 318 patients enrolled, 92 (28.9%) were positive to H1N1. Patients infected with H1N1 were mainly young adults and complained classic influenza-like symptoms. Fever was observed for a median time of 5 (IQR 3-7) days. Hospitalization occurred in 27.7% with 2% requiring intensive care unit admission: median length of hospitalization was 6 days (IQR 5-9). Pneumonia was diagnosed in 19.6% of patients. A similar proportion of lower airways involvement and of clinical complications was observed in subjects testing positive or negative for H1N1. However, patients infected with H1N1 were younger and hospitalized for a shorter period as compared to the control group (P = 0.002 and P = 0.045, respectively). Older age, asthma/chronic obstructive pulmonary disease and hypertension were associated with an increased risk of pneumonia. Viral shedding was observed for at least 1 week in 21.3% of patients. Asymptomatic infection was uncommon (1.1%). Respiratory syndromes caused by H1N1 and factors associated with disease severity were investigated and compared to influenza-like illnesses of other origin. Such findings might contribute to improve clinical and epidemiological management of the disease.