Subgingival periodontal pathogens in Down syndrome children without periodontal breakdown. A case-control study on deciduous teeth.

AIM: Down syndrome is the most common form of aneuploidia compatible with a long survival. The affected subjects are more susceptible to severe early-onset periodontal disease and show a lower risk to develop dental caries than the non-affected population. This study investigated the prevalence of periodontal pathogens in the subgingival plaque of deciduous teeth in children with Down syndrome without signs of periodontal breakdown. METHODS: Thirty children suffering from Down syndrome and 46 matched healthy subjects were studied. A total of 228 subgingival plaque samples from deciduous teeth were separately collected and evaluated by polymerase chain reaction assays. CONCLUSION: In absence of periodontal impairment, Down syndrome children display a clear presence of periodontal pathogens already in the deciduous dentition. The hypothesis of an intrinsic predisposing condition is here supported.

Bloodstream infection caused by KPC-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam: epidemiology and genomic characterization.

OBJECTIVES: The aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection. METHODS: We collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing. RESULTS: During the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime-avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime-avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of blaKPC gene. CONCLUSIONS: Our results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the blaKPC gene or porin deficiency associated with higher blaKPC copy number and is also related to the meropenem/vaborbactam resistance.

No correlation between statin exposure and incident diabetes mellitus in HIV-1-infected patients receiving combination antiretroviral therapy.

OBJECTIVES: Recent clinical studies and one meta-analysis have shown a modest but significant increase in the incidence of diabetes mellitus associated with statin exposure, so this correlation was investigated in a cohort of HIV-positive subjects. METHODS: A retrospective cohort study including adult HIV-1-infected patients followed at our Clinic of Infectious Diseases between 2007 and 2014 was performed. RESULTS: We assessed 3170 HIV-positive patients with a median follow-up of 5.2 years. The incidence of diabetes mellitus was 1.2 per 100 person-years and it was not significantly associated with the prescription of statins [hazard ratio (HR) 1.09 per year of statin exposure; 95% confidence interval (CI) 0.7-1.49; P = 0.067], while it was associated with older age, chronic hepatitis C, antiretroviral-naïve vs. antiretroviral experienced condition, high body mass index, and high serum concentration of triglycerides. CONCLUSIONS: In our study, a higher risk of diabetes mellitus was not associated with statin treatment, but with some traditional risk factors.

HIV-1 coreceptor usage in paired plasma RNA and proviral DNA from patients with acute and chronic infection never treated with antiretroviral therapy.

Although an independent evolution of viral quasispecies in different body sites might determine a differential compartmentalization of viral variants, the aim of this paper was to establish whether sequences from peripheral blood mononuclear cells (PBMCs) and plasma provide different or complementary information on HIV tropism in patients with acute or chronic infection. Tropism was predicted using genotypic testing combined with geno2pheno (coreceptor) analysis at a 10% false positive rate in paired RNA and DNA samples from 75 antiretroviral-naïve patients (divided on the basis of avidity index into patients with a recent or long-lasting infection). A high prevalence of R5 HIV strains (97%) was observed in both compartments (plasma and PBMCs) in patients infected recently. By contrast, patients with a long-lasting infection showed a quite different situation in the two compartments, revealing more (46%) X4/DM in PBMCs than patients infected recently (3%) (P = 0.008). As- a knowledge of viral strains in different biological compartments might be crucial to establish a therapeutic protocol, it could be extremely useful to detect not only viral strains in plasma, but also viruses hidden or archived in different cell compartments to better understand disease evolution and treatment efficacy in patients infected with HIV.

HIV-1 DNA proviral load in treated and untreated HIV-1 seropositive patients.

As proviral human immunodeficiency virus type 1 (HIV-1) DNA can replenish and revive viral infection upon activation, its detection might offer significant therapeutic information, complementing the input provided by plasma RNA determination in the follow-up of infected individuals. A selected group of acutely infected subjects was studied to verify both total and 2-long terminal repeat (2-LTR) DNA proviral load during the acute phase of infection and thereafter. Patients were divided in two sex- and age-matched groups: 19 naive individuals who did not receive antiretroviral therapy during the observation period and 20 subjects treated according to current guidelines. Total and 2-LTR HIV-1 DNA proviral load, in addition to RNA viral load and CD4 cell count, were determined in peripheral blood mononuclear cells (PBMC) at baseline, 6 and 12 months after the first sampling. Total and 2-LTR HIV-1 DNA proviral load exhibited no significant variation at any time in the naive patients (total HIV-1 DNA ranging from 896 + or - 731 to 715 + or - 673 copies/10(5) PBMC and 2-LTR HIV-1 DNA ranging from 94 + or - 105 to 65 + or - 44 copies/10(5) PBMC), whereas a significant reduction in both total HIV-1 DNA (ranging from 997 + or - 676 to 262 + or - 174 copies/10(5) PBMC) and 2-LTR HIV-1 DNA proviral load (ranging from 116 + or - 55 to 26 + or - 35 copies/10(5) PBMC) was detected in highly active antiretroviral therapy (HAART) patients, together with a CD4(+) T cell count increase and RNA load decrease. HAART negatively affects both the labile HIV burden and the integrated proviral DNA, at least in the initial period of successful treatment, suggesting that quantification of HIV-1 DNA proviral load may be an important parameter in monitoring HIV infection.

Discordant resistance interpretations in multi-treated HIV-1 patients.

The routine determination of drug resistance has become an important part of the clinical management of HIV-1 infected patients. Plasma samples from 130 individuals treated for at least 1 year with multiple NRTIs and NNRTIs were tested for the presence of mutations correlated to drug resistance. Since interpretation criteria represent a crucial point for virologists and clinicians, often complicated by the presence of novel and/or complex mutations patterns, we analyzed results interpreted by TruGene HIV-1 (Visible Genetics, Toronto, Ontario, Canada) and VirtualPhenotype (Virco, Mechelen, Belgium). A high degree of concordance was found for NNRTIs whereas NRTIs interpretation was highly discrepant. Since different approaches to monitoring resistance reflect different interpretation of results, the prediction of drugs resistance from a given HIV sequence might be contradictory and requires accurate standardization and unique interpretative rules.

An imported case of adult T cell leukemia in a HTLV-I-infected patient in Italy.

In this study we report the case of an acute form of ATL in a HTLV-I-infected Nigeria-born 27-year-old female prostitute living in Italy from February, 2001. The presence of HTLV-I infection was demonstrated by the detection of serum antibody to HTLV-I by immunoenzymatic assay and western blot analysis. In addition, the presence of HTLV-I proviral DNA was confirmed by a hemi-nested PCR in a sample of peripheral blood mononuclear cells. From an epidemiological point of view, it is important to report new cases of imported ATL, as it may explain the otherwise untraceable origin of some rare and apparently autochthonous cases of ATL in non-endemic areas.

Conflicting interpretations of the prevalence of mutations associated with drug resistance in antiviral naïve HIV-1 patients with acute and chronic infection.

The routine determination of drug resistance in newly HIV-1 infected individuals records a potential increase in transmissions of drug-resistant variants. Plasma samples from 38 individuals classified as newly infected (seroconversion time <12 months) and twenty four individuals with an established infection (seroconversion time ranging from 3 to 10 years) were analyzed for the presence of mutations by Trugene HIV-1 genotyping assay and Virtual phenotype. Results on the newly infected and the chronically infected individuals showed a limited number of relevant mutations associated with substantial resistance to reverse transcriptase and protease inhibitors. In particular, three patients (4.8%) carried viral major mutations (T69D and M41L) associated with resistance to reverse transcriptase inhibitors, whereas only one showed the presence of M46L, which is correlated with partial resistance to some protease inhibitors. The clinical interpretation based on different approaches to monitor resistance showed that the Virconet interpretation was less grave than Trugene, suggesting that these interpretations need standardization for the currently used sequencing methods and that they may be associated with different outcomes when eventually are used.

Drug failure during HIV-1 treatment. New perspectives in monitoring drug resistance.

Since the discovery of 3'-azido-3'deoxthymidine (zidovudine) as an effective antiretroviral agent against human immunodeficiency virus type 1 (HIV-1), drug therapy has been widely used in the treatment of AIDS. To date, new combination therapies have significantly altered the longterm prognosis for HIV-infected patients showing a reduction of plasma viral load, associated with clinical and immunological recovery. Nevertheless, in various circumstances treatment can fail for several reasons, such as patient noncompliance with the therapeutic regimen, suboptimal antiviral drug concentrations, drug pharmacokinetics, and virus resistance to one or more drugs. Virus drug resistance is the most important factor contributing to the failure of antiretroviral therapy. Since some evidence indicates that viral resistance and treatment failure are closely linked, this brief review explores the routine determination of drug resistance and its importance to shed more light on the meaning of mutations correlated to drug resistance.

Prevalence and virologic consequences of HIV-1 genotype mutations detected in a cohort of 161 Italian patients receiving a nelfinavir-based highly active antiretroviral therapy.

A cross-sectional study was carried out in our tertiary care hospital between January 1998 and December 2001. All 161 consecutive patients naive to nelfinavir and who had received a nelfinavir-based highly active antiretroviral therapy (HAART) of at least 24-week duration were extrapolated from the 802 adult HIV-infected subjects treated with antiretroviral therapy. All cases of virologic failure were considered and viral genotyped. Virologic failure occurred in 80 out of 161 nelfinavir-treated patients, all belonging to the experienced group. On the whole, only 11 patients (7%) developed the D30N substitution, whose 6 was in association with the N88D mutation. Among the 80 failed patients, the M184V mutation was detected in 52 (65%), while only 7 patients showed simultaneously the M184V, T215Y and K103N substitutions. In our HIV-infected population receiving a nelfinavir-based HAART, the D30N mutation has shown a low absolute frequency, while the detection of M184V substitution and the simultaneous occurrence of M184V, T215Y and K103N mutations were related to a more favorable virological response.

Selective up-regulation of functional CXCR4 expression in erythroid cells by HIV-1 Tat protein.

CXCR4 is the high affinity receptor for the SDF-1 alpha chemokine and represents the main coreceptor for HIV-1 T-tropic strains. The surface expression of CXCR4 was analysed in CD34+ haematopoietic progenitors, induced to differentiate along the erythroid or granulocytic lineages, in liquid cultures supplemented or not with HIV-1 Tat protein. At concentrations as low as 1-10 ng/ml, synthetic Tat protein significantly increased the surface expression of CXCR4 in erythroid but not in granulocytic cells. The Tat-mediated up-regulation of surface CXCR4 was accompanied by a concomitant increase of CXCR4 mRNA and total CXCR4 protein content in cells developing along the erythroid lineage after 6-10 days of culture. Moreover, addition of SDF-1 alpha (200 ng/ml) induced a significant higher rate of apoptosis in Tat-treated erythroid cells in comparison with control cells. These results demonstrated for the first time a direct positive role in haematopoietic gene regulation of Tat protein, and suggest the possible involvement of Tat in HIV-1-induced anaemia.

Development of drug resistance in HIV-1 patients receiving a combination of stavudine, lamivudine and efavirenz.

The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.

Prevalence of multiple dideoxynucleoside analogue resistance (MddNR) in a cohort of Italian HIV-1 seropositive patients extensively treated with antiretroviral drugs.

As the emergence of highly resistant virus might compromise antiretroviral regimens in HIV-1 infected patients, a constant analysis of genotypic mutations should be performed to establish the magnitude of mutation prevalence and gauge their impact in patients treated extensively with combination therapy. The frequency of multiple dideoxynucleoside analogue resistance (MddNR) was evaluated in a group of Italian HIV-1 seropositive patients who failed to respond to therapy despite a long-lasting drug treatment. Results showed the presence of one or more mutations (A62V, V75I, F77L, F116Y and Q151M) able to confer resistance to all NRTIs in a relatively high percentage (7.9%) of patients enrolled in the study. Moreover, a significantly lower HIV-1 viral replication in patients with MddNR, suggested the importance of monitoring HIV-1 subjects not only by viral load, but also by drug resistance testing, so that a correct drug regimen may be chosen.

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients.

BACKGROUND: The routine determination of drug resistance in newly HIV-1 infected individuals documents a potential increase in the transmission of drug-resistant variants. Plasma samples from twenty seven therapy naive HIV-1 infected Italian patients were analyzed by the line probe assay (LIPA) and the TruGene HIV-1 assay for the detection of mutations conferring resistance to HIV-1. RESULTS: Both tests disclosed amino-acid substitutions associated with resistance in a variable number of patients. In particular, two mutations (K70R and V118I), detectable by LIPA and by sequencing analysis respectively, revealed resistance to NRTIs in two plasma samples. At least three mutations conferring resistance to NNRTIs, not detectable by commercial LIPA, able to reveal mutations associated only with nucleoside reverse transcriptase analogues, were disclosed by viral sequence analysis. Moreover, most samples showed mutations correlated with resistance to protease inhibitors. Remarkably, a key mutation, like V82A (found as a mixture), and some "indeterminate" results (9 samples), due the absence of signal on the lines corresponding to a specific probe, was revealed only by LIPA, while a variable number of secondary mutations was detectable only by TruGene HIV-1 assay. CONCLUSION: Even if further studies are necessary to establish the impact of different tests on the evaluation of drug-resistant strains transmission, LIPA might be useful in a wide population analysis, where bulk results are needed in a short time, while sequencing analysis, able to detect mutations conferring resistance to both NRTIs and NNRTIs, might be considered a more complete assay, albeit more expensive and more technically complex.

Relationships between the presence of anti-Tat antibody, DNA and RNA viral load.

The possible relationships between the intensity of humoral response to full length Tat protein, the amount of proviral DNA reservoir in peripheral blood mononuclear cells and RNA viral load were analyzed in plasma samples obtained from a group of HIV-1 seropositive subjects, who never received any antiretroviral therapy. All HIV-1 patients showed detectable levels of serum IgG to full-length Tat by immunoenzymatic assay. We found a higher percentage of HIV-1 seropositive subjects with low levels of antibody in the presence of barely detectable proviral DNA copies (< or =10 copies/1.5x10(5) PBMCs) and a high anti-Tat antibody response accompanied by variable (from >10(1) to > or =10(3) copies/1.5x10(5) PBMCs) levels of DNA load (p=0.011). Moreover, an inverse relationship between anti-Tat antibody titers and HIV-1 RNA viral load was demonstrated HIV-1 seropositive patients. In HIV-1-infected patients, a strong humoral immune response against HIV-1 transactivating Tat protein, able to down-modulate viral replication in peripheral blood, does not seem to inhibit the number of proviral DNA molecules in peripheral blood mononuclear cells. Even though our data strongly confirm the "positive" role of anti-Tat antibody on viral replication, the persistence of significant amount of DNA viral load in peripheral blood mononuclear cells, despite high level of anti Tat antibody, suggests a more cautious approach to HIV-1 Tat-containing vaccines, able to stimulate an immune specific response to transactivating Tat protein sufficient in inhibiting circulating virus, but not completely efficient in decreasing proviral DNA integration.