DAA Treatment Failure in a HIV/HBV/HCV Co-Infected Patient Carrying a Chimeric HCV Genotype 4/1b.

Approved direct antiviral agent (DAA) combinations are associated with high rates of sustained virological response (SVR) and the absence of a detectable hepatitis C viral load 12-24 weeks after treatment discontinuation. However, a low percentage of individuals fail DAA therapy. Here, we report the case of a HIV/HBV/HCV co-infected patient who failed to respond to DAA pangenotypic combination therapy. The sequencing of NS5a, NS5b, NS3 and core regions evidenced a recombinant intergenotypic strain 4/1b with a recombination crossover point located inside the NS3 region. The identification of this natural recombinant virus underlines the concept that HCV recombination, even if it occurs rarely, may play a key role in the virus fitness and evolution.

Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study.

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017-February 2020 (pre-pandemic period) and March 2020-May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

Presence of V72I, G123S and R127K Integrase Inhibitor polymorphisms could reduce ART effectiveness: a retrospective longitudinal study.

Objectives: Structural aspects of HIV-1 integrase complex and role of integrase minor mutations and polymorphisms in ART effectiveness is still unknown. The objective of this study was to assess the 24 and 48 weeks (W) effectiveness of ART regimens in patients with Integrase Inhibitors (InSTI) minor mutations and polymorphisms receiving InSTI-based regimens.Methods: We enrolled all ART-naïve or InSTI-naïve HIV-infected patients, with a baseline InSTI genotypic resistances test between 2011 and 2016. We analyzed integrase resistance mutations using the Stanford University HIV Drug Resistance Database (HIVdb Program, version 6.3.0). The outcome was virological response at 24 and 48 W of follow up (FU) according to snapshot analysis. We defined virological failure as two consecutive HIV-RNA > 50 copies/ml, or one >1000 copies/ml. Patients were divided in those presenting InSTI minor mutations (Group 1), and those with only polymorphisms or wild type (Group 2).Results: We enrolled 83 patients. 81 patients reached 24 W of FU: 2/20 (10%) and 4/61 (6.5%) showed virological failure in Group 1 and 2 respectively. 66 patients reached 48 W of FU: 0/17 (0%) and 2/49 (4%) showed virological failure in Group 1 and 2 respectively. Interestingly, patients with polymorphisms G123S and R127K had higher risk of failure at 24 W (respectively, relative risk - RR - 36, IQR 2.1-613, p = 0.01; RR 36, IQR 2.1-613, p = 0.01) and patients with V72I had an higher risk of failure both at 24 W (RR 6.52, IQR 1.29-32.9, p = 0.02) and 48 W (RR 21.1, IQR 1.07-414, p = 0.04).Conclusions: Our study showed that the presence of V72I, G123S and R127K polymorphisms could play a role in reducing InSTI effectiveness.

Prevalence of Non-B HIV-1 Subtypes in North Italy and Analysis of Transmission Clusters Based on Sequence Data Analysis.

HIV-1 diversity is increasing in European countries due to immigration flows, as well as travels and human mobility, leading to the circulation of both new viral subtypes and new recombinant forms, with important implications for public health. We analyzed 710 HIV-1 sequences comprising protease and reverse-transcriptase (PR/RT) coding regions, sampled from 2011 to 2017, from naive patients in Spedali Civili Hospital, Brescia, Italy. Subtyping was performed by using a combination of different tools; the phylogenetic analysis with a structured coalescence model and Makarov Chain Monte Carlo was used on the datasets, to determine clusters and evolution. We detected 304 (43%) patients infected with HIV-1 non-B variants, of which only 293 sequences were available, with four pure subtypes and five recombinant forms; subtype F1 (17%) and CRF02_AG (51.1%) were most common. Twenty-five transmission clusters were identified, three of which included >10 patients, belonging to subtype CRF02_AG and subtype F. Most cases of alleged transmission were between heterosexual couples. Probably due to strong migratory flows, we have identified different subtypes with particular patterns of recombination or, as in the case of the subtype G (18/293, 6.1%), to a complete lack of relationship between the sequenced strains, revealing that they are all singletons. Continued HIV molecular surveillance is most important to analyze the dynamics of the boost of transmission clusters in order to implement public health interventions aimed at controlling the HIV epidemic.

Extensively drug-resistant Acinetobacter baumannii isolated from intensive care units in northern Italy: a genomic approach to characterize new sequence types.

Aim: This study aims to characterize clinical strains of Acinetobacter baumannii with an extensively drug-resistant phenotype. Methods: VITEK® 2, Etest® method and broth microdilution method for colistin were used. PCR analysis and multilocus sequence typing Pasteur scheme were performed to identify bla-OXA genes and genetic relatedness, respectively. Whole-genome sequencing analysis was used to characterize three isolates. Results: All the isolates were susceptible only to polymyxins. blaOXA-23-like gene was the only acquired carbapenemase gene in 88.2% of the isolates. Multilocus sequence typing identified various sequence types: ST2, ST19, ST195, ST577 and ST632. Two new sequence types, namely, ST1279 and ST1280, were detected by whole-genome sequencing. Conclusion: This study showed that carbapenem-resistant A. baumannii isolates causing infections in intensive care units almost exclusively produce OXA-23, underlining their frequent spread in Italy.

Role of HPV DNA, HPV mRNA and cytology in the follow-up of women treated for cervical dysplasia.

The aim of this study was to assess the role of cytology, human papilloma virus (HPV) DNA and human papilloma virus messenger RNA (HPV mRNA) assays in detecting cervical intraepithelial neoplasia grade 2+ (CNi 2+) (recurrences/persistence) during the follow-up of women after treatment of cervical intraepithelial lesion. This cross-sectional study was performed among 43 women treated for cervical intraepithelial neoplasia (CIN) between January 2014 and January 2017 at the Department of Obstetrics and Gynecology of Spedali Civili's Hospital, Brescia, Italy. Pap smear and cervical samples for HPV tests were collected during the follow-up visit. Furthermore, colposcopy was always performed in order to find out the persistence/recurrence of the disease. A cervical biopsy was collected when necessary. Cervical samples obtained were tested for HPV DNA using the INNO-LiPa HPV assay and for HPV mRNA using the APTIMA assay. The mean age of enrolled women was 42.5 years. Among the treated patients, more than 50% of women revealed the absence of high risk HPV DNA and HPV mRNA. We found the persistence of the disease cervical intraepithelial neoplasia grade 2 (CIN 2) only in one woman. The sensitivity of cytology, HPV DNA and HPV mRNA in detecting disease was satisfactory (100%), while the specificity was quite different for the three tests: 64.2, 52.4 and 78.9%, respectively. The HPV mRNA test has higher specificity with respect to cytology and HPV DNA, avoiding the referral to unnecessary colposcopy with an improvement of costs/benefits for healthcare system. However, given the small size sample, this study should be considered as a pilot for future larger studies.

Fulminant septic shock caused by Capnocytophaga canimorsus in Italy: Case report.

Capnocytophaga canimorsus infection was recently recognized as a zoonosis. We report the first case of fulminant septic shock in Italy caused by this pathogen. The patient, with a history of splenectomy, died at the main hospital in Brescia with a presumptive diagnosis of sepsis. PCR and sequencing on post mortem samples confirmed C. canimorsus as a causative organism. Our purpose is to alert medical professionals to the virulence of C. canimorsus in asplenic and immunocompromised patients.

Prevalence of Integrase Strand Transfer Inhibitors Resistance Mutations in Integrase Strand Transfer Inhibitors-Naive and -Experienced HIV-1 Infected Patients: A Single Center Experience.

Integrase strand transfer inhibitor (InSTI) resistance rates are low. However, genotypic resistance test (GRT) is not routinely performed in many centers. The aim of this study is to evaluate the prevalence of InSTI-related mutations in our large cohort. We examined all integrase GRTs performed as part of routine clinical practice at Spedali Civili General Hospital, University of Brescia from 2011 to 2016. Analysis was performed through the Stanford HIV Drug Resistance Database. A total of 341 patients were included. Genotypic resistance assays were performed in naive (48), ART-experienced but InSTI-naive (114), and both ART-experienced/InSTI-experienced (179) patients. No major resistance-associated mutations (RAMs) were detected in patients never exposed to InSTIs. Of 179 samples from patients exposed to InSTIs (mostly to raltegravir [RAL]), the overall prevalence of major RAMs was 11.7%. Among them, 10 harbored N155H, 4 Q148H, 2 Q148R, 2 Y143C/S, and 2 T66A/I/T, respectively. A novel mutation at a recognized resistance site (E92K) was identified in one RAL-experienced patient. The overall prevalence of InSTI mutations in our cohort was low, particularly in naive patients indicating no transmitted RAMs, although in InSTIs-experienced patients the rate of RAMs was high (11.7%). We support an implementation of surveillance of InSTI resistance.

Detection and molecular characterization of enteric viruses in children with acute gastroenteritis in Northern Italy.

Enteric viral infections are a major concern for public health, and viral acute gastroenteritis is the principal cause of pediatric morbidity and mortality worldwide, mostly in developing countries. The purpose of this study was to determine the prevalence of different enteric viruses detected in a pediatric population with acute gastroenteritis symptoms, and to characterize the strains detected. Stools were collected from children, aged from 2 months to 15 years old, admitted to one of the main hospitals of Northern Italy, between November 2015 and October 2016. Stools were tested for nine enteric viruses (adenovirus, rotavirus A, norovirus, astrovirus, sapovirus, enterovirus, parechovirus, bocavirus and aichivirus) by molecular methods. Furthermore, rotavirus, norovirus and adenovirus were deeply characterized by nucleotide sequencing and phylogenetic analysis. A total of 151 out of 510 (29.6%) stools analyzed resulted positive for at least one of the enteric virus investigated. The most common virus detected was rotavirus A (53/151, 35.1%), followed by norovirus (39/151, 25.8%), adenovirus (35/151, 23.1%), sapovirus (9/151, 6%), enterovirus (5/151, 3.3%), astrovirus (5/151, 3.3%), parechovirus (4/151, 2.6%) and bocavirus (1/151, 0.6%). Aichi virus was not detected in any sample. Co-infections were detected in 12 out of 510 faecal samples (2.3%). These data improved the knowledge of the enteric viruses circulating in children in Northern Italy. In fact, besides rotavirus, adenovirus and norovirus, several viruses circulated across the whole year in the pediatric population object of this study. The introduction of specific viral diagnosis in our clinical setting will improve patient care by reducing unnecessary use of antibiotics addressing the right etiologic diagnosis.

Hepatitis B virus reactivation after effective sofosbuvir and ribavirin treatment in a patient with occult hepatitis B virus infection.

Reactivation of the hepatitis B virus (HBV) has been reported in patients with occult infection (OBI), i.e. HBV surface antigen (HBsAg) negative, HBV core antibody (anti-HBc) positive ± antibodies against HBsAg (anti-HBs) and detectable HBV DNA in serum or liver, receiving immunosuppressive or cytotoxic therapies. Recently, concerns have been raised regarding the risk of HBV reactivation in OBI patients treated with direct acting antiviral agents (DAAs) for chronic hepatitis C (CHC). Here we describe a case of HBV reactivation in a 72-year-old woman with OBI as a possible consequence of effective treatment with sofosbuvir (SOF) and ribavirin (Rbv) for genotype 2a/2c CHC.

Analysis of Point Mutations in the pbp2x, pbp2b, and pbp1a Genes of Streptococcus agalactiae and Their Relation with a Reduced Susceptibility to Cephalosporins.

The emergence of group B Streptococcus (GBS) isolates with reduced penicillin susceptibility (PRGBS) and their tendency to be nonsusceptible to fluoroquinolones prompted us to analyze the possible presence of amino acid mutations in penicillin-binding proteins (PBPs) (PBP2X, PBP1A, and PBP2B) from a collection of fluoroquinolone-resistant GBS isolates. We analyzed 21 GBS isolates resistant to levofloxacin. Sequence analysis of genes for PBPs was performed. The minimal inhibitory concentrations (MICs) for penicillin, ceftibuten, cefaclor, cefixime, cefotaxime, and ceftizoxime were performed by the Etest method and by broth microdilution method. The isolates were furthermore characterized by PCR-based capsular typing and analysis of surface protein genes. Genetic relatedness among the isolates was examined by multilocus sequence typing. Phylogenetic analysis of PBPs sequences was performed by Molecular Evolutionary Genetics Analysis software (MEGA7). All isolates were susceptible to penicillin, even if different mutations were detected in all PBPs in most of the isolates (12/21, 57%). However, we observed a reduced susceptibility to cefixime in seven isolates and to cefaclor in six isolates. These PSGBS isolates shared an I377V mutation in PBP2X and a T145A mutation in PBP1A. Most of the isolates belongs to the clonal complex 1, has serotype III and rib as surface protein. The results of phylogenetic comparative analysis show that several genetic lineages of our isolates with reduced susceptibility to cefixime/cefaclor have been independently emerging through the accumulation of mutations in their pbp genes, especially in pbp1a. If the MICs of penicillins and cephalosporins for GBS increase, careful epidemiological surveillance on this issue is recommended.

Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends.

Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infection in adolescents in Northern Italy: an observational school-based study.

BACKGROUND: We carried out a study to evaluate the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae genital infections in school-based adolescents in Northern Italy. METHODS: Systematic screening for C. trachomatis and N. gonorrhoeae genital infection was performed in 13th grade students in the province of Brescia, an industrialized area in Northern Italy. Student filled in a questionnaire on sexual behaviour and provided a urine sample for microbiological testing. RESULTS: A total of 2,718 students (mean age: 18.4 years; 59.1% females) provided complete data (62.2% of those eligible). Overall 2,059 students (75.8%) were sexually active (i.e. had had at least one partner), and the mean age at sexual debut was 16.1 years (SD: 1.4). Only 27.5% of the sexually active students reported regular condom use during the previous 6 months, with higher frequency in males than in females (33.8% vs 24.2%). No case of N. gonorrhoeae infection was detected, while C. trachomatis was found in 36 adolescents, with a prevalence of 1.7% (95% CI: 1.2-2.4) among sexually active students, and no statistical difference between females and males (1.9 and 1.4%, respectively). Inconsistent condom use (odds ratio, OR = 5.5) and having had more than one sexual partner during the previous 6 months (OR = 6.8) were associated with an increased risk of Chlamydia infection at multivariate analysis. CONCLUSION: The prevalence of C. trachomatis infection among sexually active adolescents in Northern Italy was low, despite a high proportion of students who engage in risky sexual behaviour. No cases of N. gonorrhoeae infection were identified.

Characterization and antibiotic susceptibility of Streptococcus agalactiae isolates causing urinary tract infections.

Streptococcus agalactiae (GBS) has been implicated in urinary tract infections but the microbiological characteristics and antimicrobial susceptibility of these strains are poorly investigated. In this study, 87 isolates recovered from urine samples of patients who had attended the Spedali Civili of Brescia (Italy) and had single organism GBS cultured were submitted to antimicrobial susceptibility testing, molecular characterization of macrolide and levofloxacin resistance, PCR-based capsular typing and analysis of surface protein genes. By automated broth microdilution method, all isolates were susceptible to penicillin, cefuroxime, cefaclor, and ceftriaxone; 80%, 19.5% and 3.4% of isolates were non-susceptible to tetracycline, erythromycin, and levofloxacin, respectively. Macrolide resistance determinants were iMLS(B) (n=1), cMLS(B) (n=10) and M (n=5), associated with ermTR, ermB and mefA/E. Levofloxacin resistance was linked to mutations in gyrA and parC genes. Predominant capsular types were III, Ia, V, Ib and IX. Type III was associated with tetracycline resistance, while type Ib was associated with levofloxacin resistance. Different capsular type-surface protein gene combinations (serotype V-alp2, 3; serotype III-rib; serotype Ia-epsilon) were detected. A variety of capsular types are involved in significant bacteriuria. The emergence of multidrug resistant GBS may become a significant public health concern and highlights the importance of careful surveillance to prevent the emergence of these virulent GBS.

Chlamydia trachomatis genotypes in school adolescents, Italy.

Chlamydia trachomatis genogroups using ompA and multilocus sequence typing (MLST) were determined in consecutive isolates from school students aged 18 or older in the district of Brescia, Italy, 2012-2013. Among 40 samples, 4 ompA genovars and 18 STs were identified. Genovar E predominated (70 %) including five STs derived from ST59 (29 % of all isolates). This study, combining ompA and MLST typing of C. trachomatis school teenagers, suggests limited mixing and sexual interchange in this population.

Clinical course of chronic hepatitis B patients receiving nucleos(t)ide analogues after virological breakthrough during monotherapy with lamivudine.

Little is known about the optimal management of patients with chronic hepatitis B (CHB) who develop drug resistance. The aim of this study was to investigate the effectiveness of different drug regimens in chronically HBV-infected patients. HBV viral load was determined using a bDNA assay and the substitutions in HBV-DNA were studied by polymerase sequencing test. The study involved 38 patients who experienced a therapeutic failure to lamivudine (LAM). The sequential treatments used were: LAM + adefovir (ADV), LAM + tenofovir (TDF), entecavir (ETV) monotherapy, ADV monotherapy and TDF monotherapy. Similar activity against HBV replication was observed with all drug regimens. Of the patients treated with LAM, 44% developed resistance mutations. The rt M204I mutation was observed more frequently. Sequential ADV add-on LAM and TDF therapy induced the appearance of resistance in 3/18 (16.6%) and in 1/8 (5.5%) treated patients, respectively. Genotype D was the most prevalent (78.9%), followed by genotype A (13%), genotype E (5.2%) and genotype C (2.6%). Our study showed that baseline serum HBV DNA is an important predictor of virologic response and that virologic breakthrough is significantly associated with the insurgence of genotypic resistance.

Chlamydia trachomatis conjunctivitis in a male teenager: a case report.

An 18 year old man was seen at a Sexually Transmitted Infections (STIs) clinic for counselling and treatment of Chlamydia trachomatis genital infection which had been diagnosed during a screening survey of high school students. For two months he had reported conjunctival hyperaemia, increased tearing, itching, and mucopurulent secretions, predominantly on the left eye. His ophthalmologist had made a diagnosis of follicular conjunctivitis and lower superficial punctate keratitis (left eye more than right eye), irresponsive to topical treatment. Chlamydial conjunctivitis was suspected and confirmed by a positive nucleic acid amplification test (NAAT) performed on conjunctival scraping. The patient was treated with azithromycin 1 g single dose orally and tetracycline/betamethasone eye ointment for one month. A complete resolution of symptoms was observed three months after aetiological treatment. This case highlights the need to include C. trachomatis infection in the differential diagnosis of acute or chronic follicular conjunctivitis among sexually active young individuals.