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.

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.

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.

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

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.

Rapid, specific detection of Salmonella Enteritidis in pooled eggs by real-time PCR.

An assay was developed for the specific detection of Salmonella Enteritidis in eggs with the use of an application of the fluorogenic 5' nuclease assay (TaqMan). In this assay, a segment of the gene sefA specific to Salmonella group D strains such as Salmonella Enteritidis was used. The amplification of the target gene products was monitored in real-time by incorporating a fluorescent dye-labeled gene-specific probe in the PCR reaction. This method correctly detected and distinguished Salmonella Enteritidis from nearly 50 of non-group D Salmonella and other non-Salmonella strains. Detection of the sefA gene was linear for DNA extracted from approximately 10(2) to 10(9) CFU/ml in phosphate-buffered saline and 10(3) to 10(8) CFU/ml in raw egg. In two trials, when applied to detection of Salmonella Enteritidis in homogenized egg pools and compared with conventional culture methods, the newly developed PCR method yielded a 100% correlation with results obtained by a conventional culture method. However, the PCR method required only 2 days, compared to the 5 days required by the culture method. The sensitivity of this assay was approximately less than 1 CFU/600 g of egg pool. The real-time PCR assay proved to be a rapid, highly sensitive test for detection and quantification of low concentrations of Salmonella Enteritidis in egg samples.

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.

Mutation patterns of the reverse transcriptase genes in HIV-1 infected patients receiving combinations of nucleoside and non nucleoside inhibitors.

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1 x 10(4) and 9 x 10(4) HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly (P=0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Comparison of homogenization methods for recovering Salmonella Enteritidis from eggs.

For Salmonella Enteritidis (SE) detection, shell eggs have been homogenized with stomachers, with electric blenders, and by hand massaging. However, to date, there have been no published reports addressing whether the method of homogenization affects the recovery of SE from raw eggs. Three inoculum levels (10, 126, and 256 SE cells per pool of 10 eggs) were used to conduct three experiments. The 10-egg pools were homogenized by one of four homogenization methods--mechanical stomaching, electric blending, hand massaging, and hand stirring-for 30 s. The homogenized eggs were then incubated at 37 degrees C, and SE colonies were enumerated after 24 and 48 h of incubation. After 24 h of incubation, no SE was recovered from egg samples from stomached or electrically blended pools inoculated with <10 cells, while levels of 106 CFU/ml were found for samples from whipped or hand-massaged pools inoculated with <10 cells. Similarly, after 24 h of incubation, the numbers of SE cells recovered from hand-massaged or hand-stirred egg pools inoculated with 126 cells were significantly larger than the numbers recovered from stomached or electrically blended egg pools inoculated with 126 cells. The number of SE cells recovered from samples homogenized with a blender was still significantly smaller than the numbers recovered from samples homogenized by the other three methods when the inoculum level was increased to 256 CFU per pool. However, the SE count for all samples approached 9 log10 CFU/ml after 48 h of incubation. It is concluded that the detection of small SE populations in shell egg samples could be improved with the use hand massaging and hand stirring for homogenization.

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs.

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.

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.

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.

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.

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.

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.

Standardization of the Whitten Effect to induce susceptibility to Neisseria gonorrhoeae in female mice.

Female mice (Mus musculus) frequently are used to study hormonally related differences in susceptibility to infectious organisms or response to pharmaceutical agents. Cyclical variation in hormone levels within a group of mice, however, challenges the experimental design of such studies in that it is often difficult to obtain sufficient numbers of mice in the desired phase of the estrous cycle at the time of treatment. The purpose of this work is to provide investigators with a standardized protocol for inducing estrus in mice through exposure to male urine (Whitten Effect). In addition, we demonstrate how the Whitten Effect can be used to induce susceptibility of mice to Neisseria gonorrhoeae infection. Female BALB/c mice were exposed to male urine via soiled bedding for 0, 24, 48, 72, or 96 h. The effect of exposure on the reproductive cycle was monitored by cytologic examination of vaginal smears and measurement of serum 17-b estradiol levels by using a nonradioactive immunoassay kit. In a separate experiment, mice were exposed to male-urine-soaked bedding for 0, 24, 72, or 96 h prior to intravaginal inoculation with Neisseria gonorrhoeae. Infection was monitored by using vaginal culture for 5 consecutive days. We found that the highest percentage of mice in estrus occurred among mice that were exposed to male-urine-soaked bedding for 96 h. Consistent with this finding was the demonstration that mice were more susceptible to gonococcal infection after exposure to male urine for 3 to 4 days. We conclude that exploitation of this natural murine behavioral response is a simple and inexpensive method by which estrus can be synchronized in a group of mice within a defined period of time. In addition, this protocol can be used to increase mouse susceptibility to experimental gonococcal infection.