Longitudinal sequencing of HIV-1 infected patients with low-level viremia for years while on ART shows no indications for genetic evolution of the virus

HIV-infected patients on antiretroviral therapy (ART) may present low-level viremia (LLV) above the detection level of current viral load assays. In many cases LLV is persistent but does not result in overt treatment failure or selection of drug resistant viral variants. To elucidate whether LLV reflects active virus replication, we extensively sequenced pol and env genes of the viral populations present before and during LLV in 18 patients and searched for indications of genetic evolution. Maximum likelihood phylogenetic trees were inspected for temporal structure both visually and by linear regression analysis of root-to-tip and pairwise distances. Viral coreceptor tropism was assessed at different time points before and during LLV. In none of the patients consistent indications for genetic evolution were found over a median period of 4.8 years of LLV. As such these findings could not provide evidence that active virus replication is the main driver of LLV

Influence of chronic azithromycin treatment on the composition of the oropharyngeal microbial community in patients with severe asthma

Background: This study of the oropharyngeal microbiome complements the previously published AZIthromycin in Severe ASThma (AZISAST) clinical trial, where the use of azithromycin was assessed in subjects with exacerbationprone severe asthma. Here, we determined the composition of the oropharyngeal microbial community by means of deep sequencing of the amplified 16S rRNA gene in oropharyngeal swabs from patients with exacerbationprone severe asthma, at baseline and during and after 6 months treatment with azithromycin or placebo. Results: A total of 1429 OTUs were observed, of which only 59 were represented by more than 0.02% of the reads. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria were the most abundant phyla and Streptococcus and Prevotella were the most abundant genera in all the samples. Thirteen species only accounted for two thirds of the reads and two species only, i.e. Prevotella melaninogenica and Streptococcus mitis/pneumoniae, accounted for one fourth of the reads. We found that the overall composition of the oropharyngeal microbiome in patients with severe asthma is comparable to that of the healthy population, confirming the results of previous studies. Long term treatment (6 months) with azithromycin increased the species Streptococcus salivarius approximately 5-fold and decreased the species Leptotrichia wadei approximately 5-fold. This was confirmed by Boruta feature selection, which also indicated a significant decrease of L. buccalis/L. hofstadtii and of Fusobacterium nucleatum. Four of the 8 treated patients regained their initial microbial composition within one month after cessation of treatment. Conclusions: Despite large diversity of the oropharyngeal microbiome, only a few species predominate. We confirm the absence of significant differences between the oropharyngeal microbiomes of people with and without severe asthma. Possibly, long term azithromycin treatment may have long term effects on the composition of the oropharygeal microbiome in half of the patients

Effectiveness of antiretroviral therapy and development of drug resistance in HIV-1 infected patients in Mombasa, Kenya

Access to antiretroviral therapy (ART) is increasing in resource-limited settings (RLS) and can successfully reduce HIV-related morbidity and mortality. However, virologic failure and development of viral drug resistance can result in reduced treatment options and disease progression. Additionally, transmission of resistant virus, and particularly multi-drug resistance, could become a public health concern. This study evaluated treatment success and development of ART drug resistance after short-term treatment among patients attending the Comprehensive HIV Care Centre (CCC) of Coast Province General Hospital, Mombasa, Kenya. One hundred and fifty HIV-infected individuals receiving ART were consecutively recruited to participate in the study. After determination of plasma viral load, patients with detectable viral load levels were subjected to genotypic drug resistance testing. At the time of sampling, 132 of the 150 participants were on ART for more than 6 months (median 21 months, IQR = 12–26). An efficient viral load reduction to below 50 copies/ml was observed in 113 (85.6%) of them. Of the 19 patients with a detectable viral load, sequencing of the protease (PR) and reverse transcriptase (RT) gene was successful in 16. Eleven (11) of these 16 patients were infected with a subtype A1 virus. Major PR mutations were absent, but mutations associated with drug resistance in RT were detected in 14 of the 16 patients (87.5%). High-level resistance against at least 2 drugs of the ART regimen was observed in 9/14 (64.3%). The 3TC mutation M184V and the NNRTI mutation K103N were most frequent but also the multi-drug resistance Q151M and the broad NRTI cross-resistance K65R were observed. The results of this study revealed a high rate of treatment success after short term ART in patients treated at a public provincial hospital in a RLS. Nevertheless, the observed high risk of accumulation of resistance mutations among patients failing treatment and the selection of multi-drug resistance mutations in some, remains of great concern for future treatment options and potential transmission to partners

Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART

Background: Successfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. Objectives: To develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. Study design: A qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. Results: Inter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28-84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/10(6) blood cells and showed significant correlation with the pre-ART CD4(+) T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. Conclusions: Quantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution

Characteristics and spread to the native population of HIV-1 non-B subtypes in two European countries with high migration rate

Background: Non-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete. Methods: Pol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering. Results: The majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM. Conclusions: The extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero-as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored

Decision tree for accurate infection timing in individuals newly diagnosed with HIV-1 infection

Background: There is today no gold standard method to accurately define the time passed since infection at HIV diagnosis. Infection timing and incidence measurement is however essential to better monitor the dynamics of local epidemics and the effect of prevention initiatives. Methods: Three methods for infection timing were evaluated using 237 serial samples from documented seroconversions and 566 cross sectional samples from newly diagnosed patients: identification of antibodies against the HIV p31 protein in INNO-LIA, SediaTM BED CEIA and SediaTM LAg-Avidity EIA. A multi-assay decision tree for infection timing was developed. Results: Clear differences in recency window between BED CEIA, LAg-Avidity EIA and p31 antibody presence were observed with a switch from recent to long term infection a median of 169.5, 108.0 and 64.5 days after collection of the pre-seroconversion sample respectively. BED showed high reliability for identification of long term infections while LAg-Avidity is highly accurate for identification of recent infections. Using BED as initial assay to identify the long term infections and LAg-Avidity as a confirmatory assay for those classified as recent infection by BED, explores the strengths of both while reduces the workload. The short recency window of p31 antibodies allows to discriminate very early from early infections based on this marker. BED recent infection results not confirmed by LAg-Avidity are considered to reflect a period more distant from the infection time. False recency predictions in this group can be minimized by elimination of patients with a CD4 count of less than 100 cells/mm3 or without no p31 antibodies. For 566 cross sectional sample the outcome of the decision tree confirmed the infection timing based on the results of all 3 markers but reduced the overall cost from 13.2 USD to 5.2 USD per sample. Conclusions: A step-wise multi assay decision tree allows accurate timing of the HIV infection at diagnosis at affordable effort and cost and can be an important new tool in studies analyzing the dynamics of local epidemics or the effects of prevention strategies

Evaluation of the Engineering Company΄s Performance

Import 05/08/2014Cílem této diplomové práce je analyzovat a zhodnotit výkonnost podniku LUCCO a.s. pomocí vybraných metod a na základě zjištěných výsledků navrhnout doporučení pro případné zlepšení.V teoretické části jsou popsány pojmy výkonnost a jednotlivé metody tj. SWOT analýza, BSC, a bonitní a bankrotní modely, které se aplikují v praktické části. V praktické části diplomové práce se nachází charakteristika podniku LUCCO a.s. a následně zde budou zpracovány a popsány jednotlivé metody a okomentována výsledná zjištění. V poslední části jsou doporučení pro zlepšení výkonnosti podniku.The aim of this thesis is to analyze and evaluate the performance of the company as LUCCO using selected methods and based on the findings propose recommendations for possible zlepšení.V theoretical part describes the concepts and the performance of each method, ie, SWOT analysis, BSC, and value and bankruptcy models that are applied in the practical part. In the practical part of the thesis is characteristic of the enterprise as LUCCO and then there will be processed and described various methods and discussed the resulting findings. In the last section are recommendations for improving business performance.152 - Katedra podnikohospodářskávelmi dobř

Deep sequencing of HIV-1 RNA and DNA in newly diagnosed patients with baseline drug resistance showed no indications for hidden resistance and is biased by strong interference of hypermutation

Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots▿

Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study