Invasive Candidiasis in Brescia, Italy: Analysis of Species Distribution and Antifungal Susceptibilities During Seven Years.

The aims of this study were to evaluate the epidemiology of nosocomial candidemia in a large teaching hospital in Brescia, Italy, and the in vitro antifungal susceptibility of isolates. We analyzed 196 isolates causing fungemia in patients admitted in our hospital, between January 2009 and December 2015. Strains were identified by VITEK 2 and MALDI-TOF MS. MICs were determined by Sensititre Yeast OneTM. The resistance was defined by using the revised CLSI breakpoints/epidemiological cutoff values to assign susceptibility or wild type to systemic antifungal agents. Most infections were caused by Candida albicans (60%), Candida parapsilosis (15%), Candida glabrata (12%) and Candida tropicalis (6%). The susceptibility rate for fluconazole was 96.5%. Non-Candida species isolates exhibited full susceptibilities to echinocandins according to CLSI breakpoints. Amphotericin B demonstrated excellent activity against all Candida species. Local epidemiological and antifungal susceptibility studies are necessary in order to improve empirical treatment guidelines.

Low-lipid diet reduces frequency and severity of acute migraine attacks.

BACKGROUND AND AIM: There is uncertainty regarding the prevention of migraine crises by changing the lifestyle of patients. The aim of this randomized, crossover intervention trial was to evaluate the effects of a low lipid intake on the incidence and severity of migraine crises, in comparison to a diet with moderate lipid intake. METHODS AND RESULTS: After a 2-month run-in when patients received preventive medication but were left on their habitual diet, a low-lipid or a normal-lipid diet was randomly prescribed for 3 months and thereafter diets were crossed over for the following 3 months. Headache was diagnosed based on the International Classification of Headache Disorders (IHCD) III criteria. The number and severity of attacks were assessed using a self-reported calendar. Adherence to the diet was assessed by a food frequency questionnaire. An analysis was performed on the 83 episodic or chronic migraineurs (63 female and 20 male), in the age range of 18-57 years, who completed both intervention periods. Obese subjects had a significantly higher number of attacks than those overweight or with normal body weight (24.7 ± 8, 16.3 ± 12, and 15.6 ± 11, respectively, p < 0.03) with a significant relationship between the body mass index (BMI) and the number of monthly attacks (r = 0.238, p < 0.03). The number (2.9 ± 3.7 vs. 6.8 ± 7.5, p < 0.001) and severity (1.2 + 0.9 vs. 1.7 ± 0.9, p < 0.01) of attacks significantly decreased during both intervention periods, with a significant difference in favour of the low-lipid diet. CONCLUSIONS: In this group of patients, the low-lipid diet significantly affected the number and severity of migraine attacks in comparison to a normal-lipid diet. ClinicalTrials.gov Identifier: NCT 01917474.

Emergence of the first levofloxacin-resistant strains of Streptococcus agalactiae isolated in Italy.

Of 901 group B streptococcus strains analyzed, 13 (1.4%) were resistant to levofloxacin (MICs of >32 µg/ml for seven isolates, 2 µg/ml for four isolates, and 1.5 µg/ml for four isolates). Mutations in the quinolone resistance-determining regions (QRDRs) of gyrase and topoisomerase IV were identified. A double mutation involving the Ser-81 change to Leu for gyrA and the Ser-79 change to Phe or to Tyr for parC was linked to a high level of fluoroquinolone resistance. In addition, two other mutational positions in parC were observed, resulting in an Asp-83-to-Tyr substitution and an Asp-83-to-Asn substitution. Different mutations were also observed in gyrB, with unknown significance. Most levofloxacin-resistant GBS strains were of serotype Ib and belonged to sequence type 19 (ST19) and clonal complex 19 (CC-19). Most of them exhibited the epsilon gene.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group.

PURPOSE: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification. RESULTS: The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification. CONCLUSIONS: Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

Phenotypes, genotypes, serotypes and molecular epidemiology of erythromycin-resistant Streptococcus agalactiae in Italy.

The purpose of this investigation was to analyse Streptococcus agalactiae (group B Streptococcus, GBS) isolates collected in Italy from vaginal and urine samples in respect to their clonality, distribution of virulence factors and antimicrobial resistance determinants. Three hundred and eighty-eight GBS were recovered from clinical samples. They were analysed for antibiotic resistance profiling. Erythromycin-resistant strains were further characterised by multilocus sequence typing (MLST), serotyping and the detection of alp genes of the alpha-like protein (Alp) family. GBS isolates represented 40 different sequence types (STs), grouped in five clonal complexes (CCs) and belonged to seven serotypes. Most serotype V strains (81%) possessed alp2-3; serotype Ia carried mainly epsilon, while the serotype III mainly rib. All isolates were susceptible to penicillin, whereas resistance to erythromycin was detected in 15% of isolates. Most erythromycin-resistant GBS strains were of serotype V (56.8%) and belonged to the CC-1 group (50%). Macrolide resistance phenotypes were the cMLS(B) (46.5%) and the M phenotypes (46.5%) due to the presence of ermB and mefA/E genes, respectively. These results provide data which establish a baseline for monitoring erythromycin resistance in this region and also provide an insight into the correlation among clonal types, serotypes, surface protein and resistance genes. The increased prevalence of strains that displayed the M phenotype strengthens the importance of the epidemiological surveillance of macrolide resistance in GBS, which may also represent an important reservoir of resistance genes for other species.

Heterogeneity and penetration of HIV-1 non-subtype B viruses in an Italian province: public health implications.

This study assessed changes in prevalence and distribution of HIV-1 non-subtype B viruses in Italian and immigrant patients over two decades in a province in Italy. All HIV-positive patients who underwent genotypic resistance testing were selected. Prevalence of non-subtype B viruses in 3-year periods was calculated. All sequences of non-subtype B and those provided by REGA as unassigned were analysed for phylogenetic relationships. In total, 250/1563 (16%) individuals were infected with a non-subtype B virus. Prevalence increased over time, reaching a peak (31.5%) in 2004-2006. In Italian patients, the most frequent subtypes were B (92.5%) and F1 (4%). F1 subtype was also prevalent in patients from South America (13.6%); in patients of African origin, CRF02_AG (54.9%) and G (12.3%) were the most frequent. HIV-1 non-subtype B infections in Italians were mostly found in patients who acquired HIV sexually. A phylogenetic relationship between F subtypes in Italian and representative HIV-1 sequences from Brazil was found. C subtypes in Italians were phylogenetically related to subtypes circulating in Brazil. Inter-subtype recombinants were also found in the latest years. The HIV-1 epidemic in Brescia province evolved to the point where about 1/3 patients recently diagnosed harboured non-B HIV subtypes. The distribution of HIV-1 non-B subtypes in Italian patients resembled that in South American patients and phylogenetic relatedness between some Italian and South American HIV-1 strains was found. The possible epidemiological link between these two populations would have been missed by looking only at risk factors for HIV acquisition declared by patients. The evidence of inter-subtype recombinants points to significant genetic assortment. Overall our results support phylogenetic analysis as a tool for epidemiological investigation in order to guide targeted prevention strategies.

Different sequence strains of Streptococcus agalactiae elicit various levels of cytokine production.

Group B streptococcus (GBS) is the most common cause of neonatal and obstetric sepsis and an increasingly important cause of septicemia in elderly subjects and immunocompromised patients. Our aim was to evaluate whether different genotypes of GBS may induce a different production of pro-inflammatory and anti-inflammatory cytokines. We used multilocus sequence typing to identify 71 clones isolated from asymptomatic healthy carriers and symptomatic individuals. All these clinical isolates were used to infect purified human monocytes. TNF-alpha, IL-6, IL-8 and IL-10 secretion was measured. Fifteen allelic sequence types (STs) were identified. The MLST (multilocus sequence typing) analysis grouped the bacteria into four different lineages (clonal cluster) and two of these were closely involved in the infection of symptomatic subjects: CC17 and CC19. Furthermore, CC17 and CC19 stimulated TNF-alpha, IL-6 and IL-8 production significantly more than the other lineages, while CC17 induced a decreased IL-10 production. These results suggest the existence of differences in immune response to infection with particular genotypes of GBS.

Comparison of the AMPLICOR human papillomavirus test and the hybrid capture 2 assay for detection of high-risk human papillomavirus in women with abnormal PAP smear.

Infection with human papillomavirus (HPV) is a necessary step in the progression to cervical cancer. Many methods for HPV testing are currently available, mostly developed to detect pools of HPV types. Hybrid Capture 2 (HC2) is one of the most widely used. A new PCR-based assay, the Roche AMPLICOR HPV test, has been recently developed. Both assays recognize a group of 13 HR HPV types contemporaneously. This study evaluated the performance of both methods for detecting high-grade cervical lesions as a part of management for abnormal PAP smears. The study population was composed of 213 women, all referred to colposcopy and histologic diagnosis following an abnormal PAP test. Biopsy-confirmed high-grade cervical intraepithelial neoplasia was used as a gold standard. Overall agreement was 84.9% with a kappa value of 0.6. When comparing the ability to detect moderate cervical intraepithelial neoplasia (CIN2+) and high-grade cervical intraepithelial neoplasia (CIN3+/cancer), AMPLICOR proved slightly more sensitive than HC2, a finding that is important when HPV testing is used in a triage of borderline smear results. Genotyping of discordant results showed a prevalence of LR-HPV types in HC2 positive/AMPLICOR negative samples, and a similar prevalence of HR- and LR-HPV types in AMPLICOR positive/HC2 negative samples. In conclusion, the study shows that the AMPLICOR assay is more sensitive than HC2, which makes it a valid alternative for routine clinical use.

Prevalence and distribution of single and multiple HPV infections in cytologically abnormal cervical samples from Italian women.

The prevalence of single and multiple HPV infections was assessed over a cohort of 213 women with cytological abnormalities and its association with cervical neoplasia established. Roche linear array HPV genotyping test was used to identify HPV genotypes. The most prevalent HPV genotypes in cervical cancer samples were HPV16 (61.2%), HPV52 (16.1%), HPV18 (12.9%) and HPV 31 (9.6%). Multiple HR and LR HPV infections, comprising between two and 5+ HPV types, were identified in 49.7% of samples, with a significantly lower number in severe dysplasia and cervical cancer samples (p<0.05). These results seem to indicate that detection of multiple HPV infection with HR-HPV types is not significantly better as a predictor of cervical cancer than single HR-HPV infection, though further longitudinal studies are needed to better clarify the relevance of these infections to the progression of cervical neoplasia.

Detection and genotyping of human papillomavirus in cervical samples from Italian patients.

Human papillomaviruses (HPVs) are etiological agents of cervical cancer. In order to assess the epidemiological incidence and frequency of different HPV types, we applied a polymerase chain reaction (PCR)-direct sequencing approach based on the use of MY09/MY11 primers as compared to Hybrid Capture assay. Cervical samples were taken from 1,500 women, both with normal and abnormal cytological smears, and we found an incidence of 6.6% of HPV infection in Brescia. Overall, 97 samples tested HPV-positive, yielding 18 HPV types. The four most frequent HPV types were: HPV 16, -31, -6, and -58. This approach could be used in ordinary laboratory settings for quick and reliable typing of known and novel HPVs from clinical specimens and it could also be applied to anti-cancer vaccine development.

Prevalence of drug resistance and newly recognised treatment-related substitutions in the HIV-1 reverse transcriptase and protease genes from HIV-positive patients naïve for anti-retrovirals.

The aim of this study was to assess the prevalence of genetic changes in either the HIV reverse transcriptase (RT) or protease (Pro) genes in a cohort of patients naïve for anti-retroviral therapy. Of 61 patients, 43 (70.5%) were infected with HIV strains harbouring at least one resistance-related mutation, with 41 (67.2%) harbouring newly recognised treatment-related mutations. Among the 61 patients, the prevalence of specific mutations in the RT gene was as follows: 39A, 1.6%; 43E, 1.6%; and 228H, 1.6%. The prevalence of specific mutations in the Pro gene was as follows: 11I, 1.6%; 13V, 26.2%; 35D, 19.6%; 45R, 1.6%; 58E, 1.6%; 62V, 31%; 72V, 11.4%; 72M, 6.5%; 72T, 3.2%; 75I, 1.6%; and 89M, 13%. A higher prevalence of newly recognised mutations was found in strains from patients infected through sexual practices (30/36 = 83.4% vs. 11/25 = 44%; p 0.0023; OR 10.91; 95% CI 3.14-40.39). These findings support the use of resistance testing in patients naïve for anti-retroviral therapy, and suggest that the possible impact of newly recognised treatment-related mutations on clinical outcome requires further investigation.

Polymorphism analysis of Epstein-Barr virus isolates of lymphoblastoid cell lines from patients with mycosis fungoides.

In order to determine whether there is an association between the presence of Epstein-Barr virus (EBV) and mycosis fungoides (MF) disease progression, PCR was performed to detect the EBV status of 20 MF patients; six EBV-positive patients were found. EBV variants may differ in their biological properties, such as their ability to transform cells; therefore, the ability of these variants to immortalize B cells in vitro was analysed. Six continuously growing cell lines were obtained from prolonged cultures of unstimulated peripheral blood mononuclear cells that were taken from the six EBV-positive patients with MF. In order to characterize the EBV strains, EBNA-2 and LMP-1/LMP-2 gene polymorphisms in the six cell lines were also analysed. All patients were followed up for 10 years and it was noticed that EBV-positive patients had a poor prognosis with rapid disease progression and high mortality rates, compared to EBV-negative patients. EBV may therefore constitute a co-factor that accelerates the progression of disease.

Predictors of long-term immunological outcome in rebounding patients on protease inhibitor-based HAART after initial successful virologic suppression: implications for timing to switch.

OBJECTIVE: To assess predictive factors of long-term immune restoration in patients who started protease inhibitor (PI)-based HAART and experienced virological rebound after initial complete success. METHOD: A retrospective longitudinal analysis of all HIV-infected patients who started their first PI-based HAART and reached viral load below 500 copies/mL was carried out in a large academic center in Italy. Patients were classified either as complete virologic responder (CR) or rebounders (REB) when confirmed plasma viremia was detected thereafter. Immunological outcome was the area under the curve (AUC) of the absolute CD4+ cell count change since the 8th month after treatment initiation (CD4+ T-cell AUC). Association between baseline characteristics, virological outcome, and CD4+ T-cell AUC was assessed by univariate and multivariate analysis. RESULTS: There were 374 patients who were included in the study. Mean follow-up was 30.2 months. There were 226/374 patients (60.4%) who remained CR while 148/374 (39.6%) presented at least one rebound (REB). Among REB patients, complete viral suppression was regained in 15/42 (35.7%) and 50/106 (47.1%) patients who underwent therapy changes or not, respectively. When multiple linear regression was carried out, previous nucleoside reverse transcriptase inhibitor (NRTI) experience and baseline CD4+ cell count below 350 cells/muL did not impair long-term immune restoration. The occurrence of rebound, its duration (> 18 months), and its magnitude (peak of viral load > 10,000 copies/mL) were independent negative prognostic factors. CONCLUSION: The occurrence of viral rebound is independently associated with significantly impaired long-term immunological restoration. The magnitude of viral rebound (< 10,000 copies/mL) and its duration (< 18 months) may be useful to identify those rebounding patients who may still profit from maintaining the current failing therapy if a more aggressive approach may be expected to be deleterious for tolerability reasons or lack of options.

Seroprevalence of HTLV-I and HTLV-II infection among immigrants in northern Italy.

To assess the prevalence of human T cell leukemia virus type I (HTLV-I) and 2 (HTLV-II) infection and the associated risk factors among immigrants living in Northern Italy, we surveyed 3017 open-population subjects from three geographical areas and 371 prisoners. In the open population, the overall prevalence was 0.3% for HTLV-I and 0.1% for HTLV-II, while among prisoners, HTLV-I and HTLV-II infection were detected in 1.4 and 0.8% of subjects, respectively. HTLV-I prevalence was higher in subjects with multiple sexual partners or sexually transmitted diseases. This association was significant in the open-population group and close to significance in prisoners. Multivariate analysis showed that human immunodeficiency virus (HIV) seropositivity remained significantly associated with HTLV-I infection in both targeted populations (OR: 11.2 in the open population; OR: 9.9 among prisoners), whereas sexual exposure was associated with HTLV-I seropositivity only for prisoners (OR: 14.3). No independent variable was related to HTLV-II infection.

Pilot dose-finding trial on interferon alpha in combination with ribavirin for the treatment of chronic hepatitis C in patients not responding to interferon alone.

BACKGROUND: Effectiveness of combination therapy with standard interferon alpha doses and ribavirin is far from being demonstrated in patients with hepatitis C non responders to interferon alpha monotherapy. Recent kinetic studies revealed that these doses may be suboptimal. AIMS: To find the criteria for optimisation of the interferon dose, to be used in combination with ribavirin in patients with hepatitis C non responders to interferon alpha monotherapy. PATIENTS: Sixty-three patients enrolled in a pilot controlled trial were treated for 6 months with ribavirin ([1000-1200 mg daily) and were randomised to concurrently receive interferon alpha 2b for 6 months at: 3 Million Units thrice weekly [group A (21 patients)], 5 MU thrice weekly [group B (21 patients)] and 5 million units daily [group C (21 patients)]. RESULTS: A sustained virological response was observed in: 1 patient from group A (5%), 2 patients from group B (9%) and 8 patients from group C (38%; p=0.02 vs group A; p=0.03 vs group B). Side-effects were not significantly different between the 3 groups. Multivariate analysis showed that infection by hepatitis C virus genotypes 2 or 3 and interferon alpha dosage of 5 million units daily were independent predictors of sustained response. CONCLUSIONS: These results suggest that higher interferon doses administered daily in combination with ribavirin could be more effective in those patients with hepatitis C who had not responded to interferon alone.

HCV-RNA occurrence in saliva of odontoiatric HCV seropositive patients.

BACKGROUND: HCV-RNA occurrence in saliva of patients suffering from C hepatitis induced to consider saliva as a possible diffusion mean of this disease. METHODS: Saliva and blood samples from 32 C hepatitis seropositive patients, followed for odontostomatologic problems in Odontoiatric Clinic of Brescia University were obtained. In every blood and saliva sample HCV-RNA concentration was evaluated following HCV-RNA 2.0 Assay (bDNA) Quantiplex test (Chiron), in Microbiology Institute of Brescia University. RESULTS: All patients showing HCV-RNA in serum presented virus in saliva also; two patients with negative HCV-RNA serum presented virus in saliva. In latter cases, we supposed that viral concentration in serum was under sensibility threshold of employed method. CONCLUSIONS: Saliva appears an easily and not invasively obtainable medium for epidemiological studies on HCV diffusion in humans. Its role in C hepatitis transmission, on the contrary, has not been cleared till now.

Liver fibrosis progression is related to CD4 cell depletion in patients coinfected with hepatitis C virus and human immunodeficiency virus.

A total of 204 patients with liver biopsy-proven hepatitis C virus (HCV) infection, 84 with and 120 without human immunodeficiency virus (HIV) coinfection, were studied, to evaluate variables possibly associated with the stage of liver fibrosis. All patients were injection drugs users, with a mean age of 32 years and an estimated duration of HCV infection of 12 years. Twenty-four patients (11%) had many fibrous septa with (5%) or without (6%) cirrhosis, 56 (27%) had few fibrous septa, and 124 (60%) had no fibrous septa. In all patients, an association was found between CD4 cell counts <500 cells/mm(3)and the presence of many fibrous septa (odds ratio, 3.2; P=.037), independent of HIV infection and other factors. These results suggest that HIV infection-induced CD4 depletion is independently associated with the severity of liver fibrosis in chronic HCV infection.

Liver damage and kinetics of hepatitis C virus and human immunodeficiency virus replication during the early phases of combination antiretroviral treatment.

In order to assess the relationship between human immunodeficiency virus (HIV) RNA, hepatitis C virus (HCV) RNA, CD4, CD8, and liver enzymes during combination antiretroviral therapy, these parameters were measured in 12 HIV-HCV-coinfected patients (who were naive for antiretrovirals) on the day before and 3, 7, 14, 28, 56, and 84 days after initiating the following treatments: stavudine and lamivudine in all patients, indinavir in 6 patients, and nevirapine in 6 patients. HIV RNA declined rapidly, CD4 cells increased slowly, and CD8 cells and liver enzymes were stable. HCV RNA showed a transient significant increase at days 14 and 21 (7.33+/-0.16 [mean +/- SE] and 7.29+/-0.2 log copies/mL vs. 7+/-0.2 log copies/mL at baseline; P<.05). These changes were similar in both treatment groups. A 2-fold alanine aminotransferase increase was observed in 4 of 12 patients; 4 of 4 patients showed increased HCV RNA. The relationship between HCV RNA increase and HIV RNA decrease indicates virus-virus interference. An HCV RNA increase may cause significant liver damage only in a minority of patients.