MALDI-TOF Mass Spectrometry Analysis and Human Post-Mortem Microbial Community: A Pilot Study.

Introduction: The human post-mortem microbiome (HPM) plays a major role in the decomposition process. Successional changes in post-mortem bacterial communities have been recently demonstrated using high throughput metagenomic sequencing techniques, showing great potential as a post-mortem interval (PMI) predictor. The aim of this study is to verify the application of the mass spectrometry technique, better known as MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), as a cheap and quick method for microbe taxonomic identification and for studying the PM microbiome. Methods: The study was carried out on 18 human bodies, ranging from 4 months to 82 years old and with a PMI range from 24 h up to 15 days. The storage time interval in the coolers was included in the final PMI estimates. Using the PMI, the sample study was divided into three main groups: seven cases with a PMI < 72 h; six cases with a PMI of 72−168 h and five cases with a PMI > 168 h. For each body, microbiological swabs were sampled from five external anatomical sites (eyes, ears, nose, mouth, and rectum) and four internal organs (brain, spleen, liver, and heart). Results: The HPM became increasingly different from the starting communities over time in the internal organs as well as at skin sites; the HPM microbiome was mostly dominated by Firmicutes and Proteobacteria phyla; and a PM microbial turnover existed during decomposition, evolving with the PMI. Conclusions: MALDI-TOF is a promising method for PMI estimation, given its sample handling, good reproducibility, and high speed and throughput. Although several intrinsic and extrinsic factors can affect the structure of the HPM, MALDI-TOF can detect the overall microbial community turnover of most prevalent phyla during decomposition. Limitations are mainly related to its sensitivity due to the culture-dependent method and bias in the identification of new isolates.

Campylobacter jejuni bacteremia in Italian pediatric patients with acute lymphoblastic leukemia: Report of two cases.

Infections caused by Campylobacter jejuni are rarely associated with extraintestinal complications. C. jejuni bacteremia is difficult to detect in patients with hematological malignancies undergoing chemotherapy where the choice of appropriate antibiotic treatment is extremely important. We report two cases of C. jejuni bacteremia in Italian pediatric patients affected by acute lymphoblastic leukemia (ALL). Agreeing with the most recent epidemiological data, both clinical isolates showed a typical phenotypic antimicrobial resistance patterns with combined resistance to ciprofloxacin and tetracycline. To our knowledge, this is the first report of C. jejuni isolation from the blood of ALL pediatric patients in Italy, and it provides important epidemiological information on this rare infection.

Seven years prevalence and distribution of high and low risk HPV genotypes in women living in the metropolitan area of Naples.

BACKGROUND: The objective of this work was to provide an update information on HPV age/genotype distribution by retrospectively analyzing a cohort of women living in the metropolitan area of Naples. METHODS: From January 2011 to December 2017, cervical scrape specimens from 1265 women, with abnormal cytological indication, were tested for HPV DNA. The presence and the viral genotypes were assessed by the Linear Array HPV genotyping test for the detection of 37 anogenital HPV-DNA genotypes. RESULTS: The overall prevalence of HPV infections was of 44.5% (95% CI 41.77-47.24). Among HR-HPV types, HPV-16 was the most common identified genotype, followed by HPV-31, -66, -59 and -51. As concern LR-HPV, HPV-53 resulted the most prevalent. Stratifying the study population by age, the total HPV infections showed a peak in younger women aged <23 years (58.5%), with a significative decrease by age (23-29 years, 54%; ≥ 30 years, 38.2%) (p < 0.001). CONCLUSION: We provided an HPV epidemiological analysis, highlighting the need to implement vaccination programmes and preventative screening strategies.

Dissecting the prevention of estrogen-dependent breast carcinogenesis through Nrf2-dependent and independent mechanisms.

Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with development of breast cancer. Both estrogen receptor-dependent and independent mechanisms can contribute to the carcinogenic effects of estrogens. Among them, the oxidative metabolism of estrogens plays a key role in the initiation of estradiol-induced breast cancer by generation of reactive estrogen quinones as well as the associated formation of oxygen free radicals. These genotoxic metabolites can react with DNA to form unstable DNA adducts which generate mutations leading to the initiation of breast cancer. A variety of endogenous and exogenous factors can alter estrogen homeostasis and generate genotoxic metabolites. The use of specific phytochemicals and dietary supplements can inhibit the risk of breast cancer not only by the modulation of several estrogen-activating enzymes (CYP19, CYP1B1) but also through the induction of various cytoprotective enzymes (eg, SOD3, NQO1, glutathione S-transferases, OGG-1, catechol-O-methyltransferases, CYP1B1A, etc.) that reestablish the homeostatic balance of estrogen metabolism via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent and independent mechanisms.

Distribution of Hepatitis C Virus Genotypes and Subtypes in the Metropolitan Area of Naples, Italy, in the Era of Interferon-Free Regimens.

AIMS: The objective of this work was to study the distribution of hepatitis C virus (HCV) genotypes and subtypes from 2010 to 2015 in 1,221 anti-HCV/HCV-RNA-positive specimens from patients living in the metropolitan area of Naples, since HCV genotypes and subtypes remain cornerstones in the management of chronic HCV infection even in the directly acting antivirals era. METHODS: The study was carried out on 1,221 anti-HCV/HCV-RNA-positive plasma samples collected between April 2010 and December 2015. RESULTS: Of the 1,221 patients enrolled, 633 (51.9%) were males and 588 (48.1%) were females, with a mean age of 60 ± 13 (SD) years. The most frequent HCV genotype observed was genotype 1 (68.1%; 1b in 55.3% and 1a in 9.5%); HCV genotype 2 was found in 289 samples (23.67%), genotype 3 in 6.47%, genotype 4 in only 19 samples, and only 2 samples were classified as genotype 5. The mean age of the patients with genotype 1a or 3 was lower (51 ± 12 and 49 ± 12 years, respectively) than those with genotype 1b (62 ± 11, p < 0.0001 for both) or 2 (62 ± 14, p < 0.0001 for both). CONCLUSIONS: The data from the present study suggest that HCV genotype 1b remains the most prevalent in this area, followed by genotype 2, 1a, and 3a.

Non-dermatophytic onychomycosis diagnostic criteria: an unresolved question.

Non-dermatophytic moulds (NDMs) have been increasingly recognised as causative agents of onychomycosis. The diagnosis of onychomycosis is most often obtained by microscopic observation of nail specimens where fungal elements can be detected and cultured by standard mycological techniques. Direct microscopic examination does not always result positive in NDM onychomycosis; therefore to perform a correct diagnosis, a proper mycological culture is often required. The purpose of our study was to evaluate the role of direct microscopic examination in the NDM onychomycosis diagnosis. The results show that only 57.2% of the specimens from onychomycosis patients could be properly diagnosed showing positivity to both direct microscopic examination and NDMs culture isolation in two or more subsequent inoculations, while 42.8% of analysed specimens with a negative direct microscopic examination, showed NDMs growth after three or more subsequent inoculations. The large proportion of false negatives (more than 42%) could be related to the duration of the infection and/or to the experience and skills of the personnel dedicated to specimen collection. We point out the need for thoroughly evaluating all specimens showing cultural growth in at least three subsequent medium inoculations, whatever the result of the microscopic examination, in order to reduce false-negative rates. This strategy would allow for more accurate diagnosis of this mycosis.

Immigrants living in an urban milieu with sanitation in Southern Italy: persistence and transmission of intestinal parasites.

In the current era of globalization, the massive movement of populations to developed countries causes a greater attention to neglected tropical diseases in places where such diseases are considered unusual. The present study was planned to assess the persistence of intestinal parasitosis in immigrants stably living in the urban central area of Naples (Southern Italy) and the spread of infection within households with a lifestyle similar to that of the country of origin. A total of 2150 stool samples were analysed with the FLOTAC dual technique, and 415 subjects (19.3 %) tested positive for pathogenic intestinal parasites. One hundred ninety-six subjects were randomly selected and monitored again after 1 year in order to evaluate the persistence of intestinal parasites in immigrants having access to proper sanitation. No pathogenic parasites were found in these 196 samples. A total of 482 cohabitants of 151 positive subjects were recruited to evaluate the interfamilial spread of the identified parasites. Only in 18 households were there subjects infected with the same parasite. Monitoring of parasites in stool samples of immigrants showed a decrease of almost all pathogenic species over the years. From the analysis of households, it is not possible to assert that there is a familial transmission. Our study provides evidence that the prevalence of parasitic infections in immigrants is likely related to the poor sanitary habits of the country of origin and that acquisition of new sanitary regulations, together with the administration of pharmacological treatment, limits the transmission in the households and in the local population of their destination.

Microbe-host interactions: structure and role of Gram-negative bacterial porins.

Gram negative bacteria have evolved many mechanisms of attaching to and invading host epithelial and immune cells. In particular, many outer membrane proteins (OMPs) are involved in this initial interaction between the pathogen and their host. The outer membrane (OM) of Gram-negative bacteria performs the crucial role of providing an extra layer of protection to the organism without compromising the exchange of material required for sustaining life. The OM, therefore, represents a sophisticated macromolecular assembly, whose complexity has yet to be fully elucidated. This review will summarize the structural information available for porins, a class of OMP, and highlight their role in bacterial pathogenesis and their potential as therapeutic targets. The functional role of porins in microbe-host interactions during various bacterial infections has emerged only during the last few decades, and their interaction with a variety of host tissues for adhesion to and invasion of the cell and for evasion of host-defense mechanisms have placed bacterial porins at the forefront of research in bacterial pathogenesis. This review will discuss the role that porins play in activating immunological responses, in inducing signaling pathways and their influence on antibiotic resistance mechanisms that involve modifications of the properties of the OM lipid barrier.

A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid.

BACKGROUND: Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools. OBJECTIVE: The development and assessment of a real-time RT-PCR assay to detect USUV in human samples. STUDY DESIGN: Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated. RESULTS: The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P. CONCLUSIONS: This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.