Anna Boroń-Kaczmarska

Characteristics of HIV-1 non-B subtype infections in Northwest Poland.

The number of non‐B subtype HIV‐1 infections in Europe has been increasing even though major regional differences have been observed. This trend was investigated in northwestern Poland using sequence and epidemiological data from a cohort of 102 HIV‐1‐infected patients from Szczecin, Poland. HIV‐1 subtypes were defined by phylogenetic analysis of viral reverse transcriptase‐ and protease‐partial coding regions, and results were compared with online subtyping by Standford and REGA tools. Subtype analysis using on‐line subtyping methods produced varying results if compared to phylogenesis, with concordant variant assignment obtained for 98% (100/102) of sequences by Stanford and 85% (87/102) by REGA. In the population studied, non‐B subtype infections comprised 21% of the infections and consisted of subtype D (57%, n = 12), CRF01_AE (19%, n = 4), A and C clades (9.5%, n = 2), and the CRF13_cpx recombinant isolate (4.8%, n = 1). Patients carrying non‐B subtypes were predominantly heterosexuals with high percentage (57%) of women observed in the group. All HIV‐1 non‐B women were Caucasian with majority (83%) of infections acquired in Poland; however, among 12 travelers included in the study a higher proportion of non‐B infections was noted (50%, P = 0.01). Moreover, lower baseline lymphocyte CD4 counts (P = 0.01), higher baseline HIV‐1 viremia (P = 0.08), and a more advanced stage of the disease (P = 0.03) were observed among individuals infected with non‐B subtypes. The data indicated that the proportion of HIV‐1 non‐B subtype infections was higher than previously reported in Poland consisting of a high subtype D prevalence. Furthermore, subtype D transmission occurred primarily between heterosexual Caucasian individuals from this region. J. Med. Virol. 82:1306–1313, 2010.

Sequence variants of chemokine receptor genes and susceptibility to HIV-1 infection

Genetic susceptibility to HIV infection was previously proven to be influenced by some chemokine receptor polymorphisms clustering on chromosome 3p21. Here the influence of 5 genetic variants was studied: Δ32CCR5, G(-2459)ACCR5, G190ACCR2, G744ACX3CR1 and C838TCX3CR1. They were screened in a cohort of 168 HIV-1 positive adults [HIV(+) group] and 151 newborns [control group] from northwestern Poland. PCR-RFLP was performed to screen for the variants (except for A32CCR5 polymorphism, where PCR fragment size was sufficient to identify the alleles) and then electrophoresed on agarose gel to determine fragment size. Distribution of genotypes and alleles was not significantly different between the groups except for theCCR5 polymorphisms, with the A32 allele and the (-2459)ACCR5 allele more frequent among neonates than in the HIV(+) group. No Δ32/Δ32 homozygotes were found in the HIV(+) group, but 16.1% were Δ32/wt heterozygotes. In the control group, 1.3% were Δ32/Δ32 homozygotes and 26.0% were Δ32/wt heterozygotes. Linkage between the chemokine polymorphisms was calculated using the most informative loci for haplotype reconstruction. Haplotypes containing Δ32 CCR5,190GCCR2 and 744ACX3CR1 were found to be significantly more common in the control group. This suggests an association between these haplotypes and resistance to HIV-1 infection.

Comparative effects of conjugated linoleic acid (CLA) and linoleic acid (LA) on the oxidoreduction status in THP-1 macrophages.

The aim of this study was to investigate the effect of conjugated linoleic acids (CLAs) on macrophage reactive oxygen species synthesis and the activity and expression of antioxidant enzymes, catalase (Cat), glutathione peroxidase (GPx), and superoxide dismutase (SOD). The macrophages were obtained from the THP-1 monocytic cell line. Cells were incubated with the addition of cis-9,trans-11 CLA or trans-10,cis-12 CLA or linoleic acid. Reactive oxygen species (ROS) formation was estimated by flow cytometry. Enzymes activity was measured spectrophotometrically. The antioxidant enzyme mRNA expression was estimated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was based on nonparametric statistical tests [Friedman analysis of variation (ANOVA) and Wilcoxon signed-rank test]. cis-9,trans-11 CLA significantly increased the activity of Cat, while trans-10,cis-12 CLA notably influenced GPx activity. Both isomers significantly decreased mRNA expression for Cat. Only trans-10,cis-12 significantly influenced mRNA for SOD-2 expression. The CLAs activate processes of the ROS formation in macrophages. Adverse metabolic effects of each isomer action were observed.

Increased concentration of interferon lambda-3, interferon beta and interleukin-10 in the cerebrospinal fluid of patients with tick-borne encephalitis

Tick-borne encephalitis (TBE) has a wide clinical spectrum, from asymptomatic to severe encephalitis, and host-dependent factors determining the outcome remain elusive. We have measured concentrations of pro-inflammatory/Th1 interferon-γ (IFNγ), immunomodulatory/Th2 interleukin-10 (IL-10), anti-viral type I (IFNβ) and type III (IFNλ3) interferons in cerebrospinal fluid (csf) and serum of 18 TBE patients, simultaneously genotyped for polymorphisms associated with the expression of genes IFNL3 (coding IFNλ3), IL10, CD209 and CCR5. IL-10, IFNβ and IFNλ3 were up-regulated in csf, with IFNλ3 level higher in patients with the milder clinical presentation (meningitis) than in meningoencephalitis. There was an increased serum IFNβ and a tendency for increased serum IL-10 in meningitis patients. Genotype in rs12979860 locus upstream of IFNL3 was associated with IFNλ3 expression and in rs287886 (CD209) - IL-10 expression. IL-10, IFNβ and IFNλ3 are expressed and play a protective role in TBE and their expression in TBE patients is associated with genetic polymorphisms.

HIV-1 integrase resistance among antiretroviral treatment naive and experienced patients from Northwestern Poland

BackgroundHIV integrase inhibitor use is limited by low genetic barrier to resistance and possible cross-resistance among representatives of this class of antiretrovirals. The aim of this study was to analyse integrase sequence variability among antiretroviral treatment naive and experienced patients with no prior integrase inhibitor (InI) exposure and investigate development of the InI drug resistance mutations following the virologic failure of the raltegravir containing regimen.MethodsSequencing of HIV-1 integrase region from plasma samples of 80 integrase treatment naive patients and serial samples from 12 patients with observed virologic failure on raltegravir containing treatment whenever plasma vireamia exceeded >50 copies/ml was performed. Drug resistance mutations were called with Stanford DB database and grouped into major and minor variants. For subtyping bootstrapped phylogenetic analysis was used; Bayesian Monte Carlo Marcov Chain (MCMC) model was implemented to infer on the phylogenetic relationships between the serial sequences from patients failing on raltegravir.ResultsMajority of the integrase region sequences were classified as subtype B; the remaining ones being subtype D, C, G, as well as CRF01_AE , CRF02_AG and CRF13_cpx recombinants. No major integrase drug resistance mutations have been observed in InI-treatment naive patients. In 30 (38.5%) cases polymorphic variation with predominance of the E157Q mutation was observed. This mutation was more common among subtype B (26 cases, 54.2%) than non-B sequences (5 cases, 16.7%), p=0.00099, OR: 5.91 (95% CI:1.77-22.63)]. Other variants included L68V, L74IL, T97A, E138D, V151I, R263K. Among 12 (26.1%) raltegravir treated patients treatment failure was observed; major InI drug resistance mutations (G140S, Q148H and N155H, V151I, E92EQ, V151I, G163R) were noted in four of these cases (8.3% of the total InI-treated patients). Time to the development of drug resistance ranged from 2.6 to 16.3 months with mean increase of HIV viral load of 4.34 (95% CI:1.86-6.84) log HIV-RNA copies/ml at the time of emergence of the major mutations. Baseline polymorphisms, including E157Q were not associated with the virologic failure on raltegravir.ConclusionsIn InI treatment naive patients polymorphic integrase sequence variation was common, with no major resistance mutants. In the treatment failing patients selection of drug resistance occurred rapidly and followed the typical drug resistance pathways. Preexisting integrase polymorphisms were not associated with the treatment failure.

HIV-1 Subtype D Infections among Caucasians from Northwestern Poland—Phylogenetic and Clinical Analysis

Background HIV-1 subtype D infections, which are associated with a faster rate of progression and lymphocyte CD4 decline, cognitive deficit and higher mortality, have rarely been found in native Europeans. In Northwestern Poland, however, infections with this subtype had been identified. This study aimed to analyze the sequence and clinical data for patients with subtype D using molecular phylogeography and identify transmission clusters and ancestry, as well as drug resistance, baseline HIV tropism and antiretroviral treatment efficacy. Methods Phylogenetic analyses of local HIV-1 subtype D sequences were performed, with time to the most recent common ancestor inferred using Bayesian modeling. Sequence and drug resistance data were linked with the clinical and epidemiological information. Results Subtype D was found in 24 non-immigrant Caucasian, heterosexually infected patients (75% of females, median age at diagnosis of 49.5 years; IQR: 29–56 years). Partial pol sequences clustered monophyletically with the clades of Ugandan origin and no evidence of transmission from other European countries was found. Time to the most common recent ancestor was 1989.24 (95% HPD: 1968.83–1994.46). Baseline drug resistance to nucleoside reverse transcriptase inhibitors was observed in 54.5% of cases (mutations: M41L, K103N, T215S/D) with evidence of clustering, no baseline integrase or protease resistance and infrequent non-R5 tropism (13.6%). Virologic failure was observed in 60% of cases and was associated with poor adherence (p<0.001) and subsequent development of drug resistance (p = 0.008, OR: 20 (95%CI: 1.7–290). Conclusions Local subtype D represented an independently transmitted network with probably single index case, high frequency of primary drug resistance and evidence of transmission clusters.

Genotypic tropism of antiretroviral‐treated patients with drug resistant HIV‐1

CCR5 inhibitors remain an attractive antiretroviral treatment option for HIV‐infected patients; however, tropism testing should be utilized prior their introduction. This study analyzed genotypic HIV‐1 tropisms in patients with evidence of genotypic drug resistance to antiretroviral therapies in Northwest Poland. V3 loop sequences were analyzed from plasma samples obtained from patients presenting with virologic treatment failure while on combined antiretroviral treatment and with evidence of genotypic drug resistance. Genotypic X4 and R5 tropisms were identified using the geno2pheno algorithm with a false positive rate threshold set at 10%. Clinical data for all patients examined was collected, in addition to determining the CCR5 Δ32 genotype and calculating the genotypic susceptibility score (GSS). Virologic treatment failure and the presence of drug resistant mutations were observed in 37/450 (8.4%) patients on cART (combination antiretroviral therapy) with successful tropism analysis carried out on 35 (95%) cases. In 22 (62.9%) and 13 (37.1%) cases the R5 and X4 tropisms were predicted, respectively. An association between viral X4 tropism and the M41L (P = 0.04) resistance mutation and R5 tropism and the K103N (P = 0.07) resistance mutation were observed. GSS values were lower in the group with NRTI (P = 0.01) and NNRTI resistance (P = 0.048). In the majority of the drug resistant patients, R5 tropic viruses were found. As genotypic tropism testing is easy to carry out and interpret, its use in clinical practice would be highly useful in determining the use of appropriate drug therapies. J. Med. Virol. 83:1869–1875, 2011.

Introduction of pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 variant in Polish HIV-infected patients.

Prospective pharmacogenetic screening for the human leucocyte antigen (HLA) B*5701 allele can significantly reduce the number of cases of abacavir‐related hypersensitivity among HIV‐infected patients treated with this drug. The aim of this study was to establish the frequency of the HLA B*5701 variant in HIV‐infected Poles.

Recommendations for the treatment of hepatitis C issued by the Polish Group of HCV Experts – 2016

BACKGROUND Diseases of HCV aetiology are rarely diagnosed on the basis of the clinical picture, as their course is usually asymptomatic or only mildly symptomatic for many years. Consequently, diagnosis is frequently preceded by an incidental detection of laboratory markers indicative of HCV infection. Studies conducted in Poland in recent years have shown that anti-HCV antibodies are found in 0.9-1.9% of the Polish population, depending on the study population and the methodology applied. The studies have consistently confirmed the presence of HCV-RNA in the blood, indicating active infection, at 0.6%. The figure corresponds to approx. 200,000 adult Poles who require immediate diagnosis and treatment. The number of patients diagnosed during the period of HCV therapy availability is estimated to be approx. 35,000, which is equivalent to the detection rate of 17.5% [1-3]. HCV genotype (GT) 1 is the most prevalent one among the Polish population: it is found in 80% of all infected individuals, the most common (98%) sub-genotype being GT1b. GT3 (14%) and GT4 (5%) are less common, and infections with genotypes 2, 5 and 6 may be diagnosed sporadically [4]. Around 20-40% of acute infections are thought to resolve spontaneously. Chronic HCV infection only manifests itself after many years, and one in five patients develop advanced pathological changes in the liver including cirrhosis or hepatocellular carcinoma. HCV infection also induces a number of extrahepatic syndromes, most typically cryoglobulinemia which gives rise to clinical manifestations in 5-25% of cases, and B-cell non-Hodgkin lymphoma (B-NHL) [5]. All patients with chronic HCV infection should receive treatment. The sooner the therapy is initiated, the better the outcome and the lower the cost. However, if access to therapy is restricted, priority should be given to those patients whose HCV infection, in the assessment of an infectious diseases specialist, can lead to a deterioration of the quality of life or death within a short time. The aims of treatment are to eliminate HCV infection and, consequently, to stop or reverse histological lesions, and to reduce the risk of development of hepatocellular carcinoma and prevent the spread of the infection to other people [6, 7].

Risk of all-cause mortality in HIV infected patients is associated with clinical, immunologic predictors and the CCR5 Δ32 deletion.

Objective Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era. Design Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed. Methods Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed. Results A mortality rate of 2.66 (CI 2.57–3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16–5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1–3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0–7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95–8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39–8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03–2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23). Conclusions The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender.