Prevalence of hepatitis C virus mutants resistant to protease inhibitors among Polish HCV genotype 1-infected patients.

AIM: The aim of this study was to evaluate prevalence of hepatitis C virus (HCV) harbouring mutations associated with decreased susceptibility to protease inhibitors (Boceprevir/Telaprevir) among Polish untreated patients infected with HCV genotype 1. MATERIAL AND METHOD: Population sequencing was used, sequencing data were interpreted by web based geno2pheno algorithm. A total of 91 serum samples were obtained from patients infected with HCV genotype 1, admitting Outpatient Clinics of Hospital of Infectious Diseases, Warsaw. RESULTS: Sequencing analysis of the NS3 protease catalytic domain was successful in 85 out of 91 subjects. In seventy three (85.9%) out of 85 samples wild-type HCV was detected; in 12 (14.1%) samples mutations associated with clinically observed Boceprevir/Telaprevir-decreased susceptibility were detected. SUMMARY AND CONCLUSIONS: Obtained results document the presence of HCV strains harbouring protease inhibitors (PIs) resistance-associated mutations among Polish therapy-naïve patients. The determined prevalence of drug resistant HCV variants is 14.1%. Further and continuous surveillance is necessary to estimate how preexisting and emerging drug resistance mutations influence clinical outcome in triple-therapy experienced patients.

Distribution of HCV genotypes in Poland.

UNLABELLED: Available data on prevalence of HCV genotypes in Poland are insufficient. The aim of the study was the analysis of distribution of HCV genotypes in Poland over the period of recent 10 years regarding the age of patients and the regions of the country. MATERIAL AND METHODS: Analysis of HCV genotypes in Poland was carried out between 2003 and 2012, and included 14 651 patients from 22 centers where patients with chronic viral hepatitis C are diagnosed and treated. Genotypes were analyzed in age groups (< 20 years of age, 20-40 years of age, > 40 years of age) as well as in populations of HBV and HIV co-infections. RESULTS: Genotype (G) 1 infection was demonstrated in 79.4%, G2 -0.1%, G3- 13.8%, G4- 4.9%, G6-0.09% and mixed infections in 1.6%. There was no infection with genotype 5. The highest prevalence of G1 was observed in the Lódzkie voivodship (89.2%) and the Slaskie voivodship (86.7%) while the lowest one in the Warminsko-mazurskie (62.0%) and the Podlaskie voivodships (68.2%). Genotype 3 most commonly occurs in the Warminsko-mazurskie (28.1%), and the Podlaskie voivodships (23.0%) and is least common in the Malopolskie (7.9%) and the Lódzkie voivodships (9.0%). Genotype 4 is more common in the Kujawsko-pomorskie (11.7%) and the Podlaskie voivodships (8.6%) and relatively less common in the Lubelskie (1.1%) and the Lódzkie voivodships (1.8%). Prevalence of G1 infection in 2003-2004 was 72% and increased up to 85.6% in 2011-2012, that was accompanied by decrease of G3 prevalence from 17% to 8% in this period. In HBV co-infected (n = 83), G1 infection was demonstrated in 85.5%, G3 - in 7.2%, G4 -4.8%, and mixed genotypes in 6%. Among HIV co-infected (n = 391), a much lower prevalence of G1 (33.0%) and a high of G3 (40.4%) as well as G4 (24.0%) were observed. CONCLUSIONS: There is a geographic variability of HCV genotypes prevalence in Poland. Increase of HCV G1 infections and decrease of G3 and G4 were observed in the last 10 years. Genotypes G3 and G4 occur more often in HCV/HIV co-infected than in HCV mono-infected patients.

Evolving patterns of HIV-1 transmitted drug resistance in Poland in the years 2000-2008.

The aim of the study was to determine the rate of transmission of drug resistant human immunodeficiency virus-1 (HIV-1) variants among therapy-naïve HIV positive patients in Poland in the year 2008, to compare the data with the results from the years 2000 to 2007 and to monitor patterns of HIV-1 subtypes present in Polish population and their evolution. Complete protease and part of reverse transcriptase regions were sequenced from the sera of patients directed to the laboratory for drug resistance testing. The Stanford's HIVdb program was used for the interpretation of results and subtyping. The variants scoring at least "intermediate resistance" for at least one drug were considered as resistant. The results obtained were compared to those obtained in the years 2000-2007. A total of 95 patients were enrolled in the 2008 study. Homosexual transmission of infection was documented in more than 55% of all cases. The overall prevalence of transmitted drug resistance (TDR) was 5.3% (3.9% in 2007, 5.8% in 2006, and 14.1% in the years 2002-2005). The study from the years 2000 to 2001 revealed 28.7% prevalence. Preliminary analysis of the first half of 2009 shows the ratio of 7.8%. In four (4.2%) cases drug resistance was associated with protease inhibitors class, in one case (1.1%) with resistance to non-nucleoside reverse transcriptase inhibitors class. In four cases (4.2%) non-B subtype was identified (C, G, CRF01_AE, CRF02_AG). An increase of percentage of drug resistant mutants-from 3.9% (2007) to 5.3% (2008)-was recognized. In this study, TDR was limited to single classes of antiretroviral drugs. HIV-1 subtype B prevails in Poland.

Better outcome of HBe Ag (-) than HBe Ag (+) lamivudine treatment of HBV chronically infected patients.

From May 2006 to December 2007 in Warsaw Hospital for Infectious Diseases one hundred and eight patients chronically infected HBV were treated with lamivudine. Among them 46 (42.5%) were HBeAg (+) and 62 (57.5%) HBeAg (-). HBV DNA levels were analysed in weeks 24, 48 and 72 of therapy using Real Time HBV PCR (Abbott) with a limit of detection of 28 copies/ml. Complete response for treatment was defined as HBV VL of less than 10(2) copies/ml (group A). Partial response was defined as HBV VL ranging from 10(2) to 10(5) copies/ml (group B), and treatment failure was determined by HBV VL above 10(5) copies/ml (group C). Presented results confirmed better response to lamivudine treatment in patients HBeAg (-) than HBeAg (+).

Prevalence of HBV genotypes in Central and Eastern Europe.

The importance of hepatitis B virus (HBV) genotypes for disease progression and response to interferon-alpha-based treatment is well established. While almost all patients in the Mediterranean area are infected with HBV genotype D, HBV genotype A is dominant in Northern Europe. However, the distribution of HBV genotypes is unknown for several Central and Eastern European countries. Data are described of 1313 HBsAg-positive patients recruited at 14 referral centers in eight countries. There were only very few cases of HBV genotype B, C, E, F, and H infection while HBV genotypes A and D were found in 42% and 48% of patients, respectively. Eight percent of patients had positive bands for more than one genotype using the hybridization assay. The frequency of genotype A was higher in Poland (77%) and the Czech Republic (67%) as compared to Hungary (47%), Lithuania (41%), Croatia (8%), and Germany (32%). In contrast, HBV genotype D was most frequent in Croatian, Romanian, and Russian patients with 80%, 67%, and 93% of cases, respectively. In conclusion, HBV genotype A versus D showed significantly different distribution patterns in Central and Eastern Europe which deserves consideration for national guidelines and treatment decisions.

[Transmission of HIV-1 drug resistance among newly diagnosed patients in Poland]. / Transmisja lekoopornych szczepów HIV-1 wsród osób nowo diagnozowanych w Polsce.

OBJECTIVE: HIV-1 drug resistance is becoming a growing concern. It is estimated that one out of ten newly diagnosed persons in Europe acquires HIV drug resistant strain. The aim of this study was to determine the transmission of drug resistance and identify the resistance patterns among naïve patients in Poland. METHODS: The patients were asked to complete a brief questionnaire concerning demographic and epidemiological data. Viral load and CD4/CD8 counts were detemined before drug resistance testing. The sequencing assay was performed according to manufacturer's protocol. MAIN OBSERVATIONS: In the analysed cohort 14.7% of patients acquired HIV-1 drug resistant strains; further 9.5% were infected with strains with "possibly lowered susceptibility". RESULTS: In all cases resistance to single class of antiretroviral drugs were identified. In the class of PIs resistance to NFV was the most common. The rates of drug resistance among NNRTIs were almost the same--about 5%. In the NRTI class the resistance to AZT and d4T was the most frequent. HIV-1 subtype B was identified in 88.8% of cases. CONCLUSIONS: The results of this study document high transmission rate of drug resistance in Poland and justify the necessity of common DR testing in our country.

[Results of 48 weeks lamivudine treatment of patients with chronic hepatitis B and HBeAg (-)]. / Ocena skutecznosci leczenia lamiwudyna przez 48 tygodni pacjentów z przewleklym wirusowym zapaleniem watroby typu b HBeag(-).

Lamivudine is an oral nucleoside analogue with strong antiviral activity against hepatitis B virus. The HBeAg-negative patients tend to have lower serum HBV viral loads when compared to HBeAg-positive patients, but may develop liver disease. The objective of this study was to analyse the efficacy of lamivudine treatment of chronic hepatitis B patients negative in HBe antigen (HBe-Ag-negative). 102 patients were treated in Hospital of Infectious Diseases in Warsaw in the years 2001-2002. Patients were treated for 48 weeks with 100 mg lamivudine once daily (50 mg in case of renal failure). The end point of therapy of patients with chronic HBV infection negative for e antigen is more difficult to determine than for HBeAg-positive patients because HBeAg seroconversion marker cannot be applied. The only useful markers of therapy efficiency are the supression of HBV DNA replication and normalization ofALT activity level. Results at the end of therapy: normalization of ALT activity was observed in 48,7% patients, inhibition of viral replication was detected in 65,6% patients. The results are comparablewith known randomized clinical trials.

[Results of 48 weeks lamivudine treatment of patients with chronic hepatitis B and HBeAg (+)]. / Ocena skutecznosci leczenia lamiwudyna przez 48 tygodni pacjentów z przewleklym wirusowym zapaleniem watroby typu B HBeAG (+).

Chronic hepatitis B is an important public health problem worldwide. In Poland the incidence rate decreased from 40,0 (in the year 1990) to 3,86 (in 2004) per 100,000 inhabitants. The goal of anti-chronic hepatitis B therapy is to prevent the progression of liver disease to cirrhosis which may effect in development of liver failure or HCC. The aims of treatment are: reduction of HBV viral load and normalization of ALT activity. Among HBeAg positive patients important marker is the loss of e antigenemia followed by seroconversion to anti-HBe positivity. Lamivudine is an oral nucleoside analogue with strong antiviral activity against hepatitis B virus. The objective of this study was to analyse the efficacy of lamivudine treatment of chronic hepatitis B patients positive for hepatitis B e antigen (HBeAg). 224 patients were treated in Hospital of Infectious Diseases in Warsaw in the years 2001-2002. Patients were treated for 48 weeks with 100 mg lamivudine once daily (50 mg in case of renal failure). Results obtained at the end of therapy: loss of HBeAg was observed in 33,4% and seroconversion to anti-HBe in 15,1% patients, normalization of ALT activity was noticed in 53,4% patients, inhibition of HBV DNA replication was observed in 37,9% patients. The results are comparable with known randomized clinical trials.

[Study of the efficacy of combined therapy with interferon alfacon-1 and ribavirin for chronic hepatitis C]. / Ocena skutecznosci leczenia skojarzonego interferonem alfacon-1 i rybawiryna pacjentów z przewleklym wirusowym zapaleniem watroby typu c.

The results of combined interferon alfacon-1 and ribavirin therapy of 94 patients with chronic hepatitis C were analyzed. Complete data, including sustained viral response (SVR), were obtained in 88 patients. 46.8% of them achieved SVR. The most important factor influencing SVR, was the presence of HCV RNA in serum at weeks 12 and 24 of therapy. SVR in these cases was achieved in 14.3% and 0%, respectively. Eight patients discontinued therapy due to adverse events. Most frequent were depressive reactions due to interferon (3 cases), and severe anemia due to ribavirin (2 cases). 37% of patients developed thyroiditis, significantly more frequent in women (27 versus 9).

[Comparison of early virologic response among patients with chronic hepatitis C infected with genotype non 2/3 treated with pegylated interferon alfa-2B and ribavirin in dependence with hepatic fibrosis stages]. / Porównanie wczesnej odpowiedzi wirusologicznej w zaleznosci od zaawansowania wlóknienia u pacjentów z przewleklym wirusowym zapaleniem watroby typu c zakazonych genotypem innym niz 2 i 3 leczonych pegylowanym interferonem alfa-2B i rybawiryna.

Early virologic response (EVR) depending on various hepatic fibrosis was analyzed at 12 week of pegylated interferon alfa-2b (Pegintron, 12 KD) with ribavirin treatment among chronic hepatitis C patients (pts) infected with genotype non 2/3. The A group composed 29 pts. They were of staging 0 and grading 1. The group B composed 47 pts of staging 1, C 33 pts of staging 2, D 35 pts of staging 3 and 4. Liver biopsies were analyzed according to the Scheuer's and Knodell's scores. Early virologic response (ERV) was defined as decrease of VL >2 log or undetectable HCV RNA. Viral load (VL) was determined with HCV RNA Assay and CA HCV Monitor Test (Roche Diagn. Sys.). The EVR rates for the A, B, C, D groups were as follow: 86,2% (25/29), 80,9% (38/47), 75,8% (25/33) and 60% (15/25), respectively.

Peginterferon alfa-2a and peginterferon alfa-2b combined with ribavirin in patients with genotype 1 chronic hepatitis C: results of a prospective single-centre study.

PURPOSE: This prospective, randomized, single-centre study compared peginterferons alfa-2a and alfa-2b, combined with ribavirin, in treating patients infected with hepatitis C virus (HCV) genotype 1. MATERIAL/METHODS: Hundred-and-one patients received 48 weeks of open-label treatment with peginterferon alfa-2a (180 µg/week) and 111 patients received peginterferon alfa-2b (1.5 µg/kg/week). All patients received the same dose of ribavirin 1000/1200 mg/day, depending on weight. The primary efficacy endpoint was sustained virologic response (SVR), defined as undetectable HCV RNA (<50 IU/mL) 24 weeks after the end of treatment. RESULTS: Early virologic response (EVR), defined as at least 2 log10 IU/mL reduction of viral load at 12 weeks, was more common in patients treated with peginterferon alfa-2a (88% vs. 74.8%; p=0.04). However, the difference in SVR was not statistically significant (49.5% vs. 44.1%; p=0.43). CONCLUSIONS: Peginterferon alfa-2a treated patients were also more likely to be HCV RNA negative at the end of treatment (67.3% vs. 57.7%), but this difference did not reach statistical significance. Multivariate logistic regression analysis found that SVR was associated with low fibrosis stage (F1-2 by Scheuer; p=0.001) and low serum HCV RNA level (<400,000 IU/L; p=0.023). While both forms of peginterferon showed similar efficacy as measured by SVR, use of peginterferon alfa-2b could lower the number of patients receiving unnecessary treatment beyond 12 weeks.

Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival.

IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02(+) subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)(+) regulatory T (T(reg)) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of T(reg) cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.

Modern diagnostics of Chlamydia trachomatis infections.

Chlamydia trachomatis (C. trachomatis) is the most common agent of sexually transmitted infections. The clinical spectrum of the disease ranges from urethritis to infertility in women and to trachoma. Intracellular localisation of the pathogen creates a challenge for routine diagnostics. In this review possible diagnostic tests have been presented, varying from classic cell culture analysis and serodiagnostics (Enzyme-linked Immunoassays, Indirect Immunofluorescence) to the most sophisticated nucleic acid analyses (hybridisation, Polymerase Chain Reaction, Transcription Mediated Amplification, Ligase Chain Reaction), Advantages and disadvantages of the leading tests are discussed. Possible reasons of false positive as well as false negative results of genetic testing are presented.

Infection of primary human macrophages with hepatitis C virus in vitro: induction of tumour necrosis factor-alpha and interleukin 8.

Hepatitis C virus (HCV) has been reported to replicate in monocytes/macrophages in infected patients. However, it is unclear whether macrophages are susceptible to infection in vitro and whether such an infection is consequential. Sera from 26 HCV-infected patients were incubated with primary human macrophages collected from healthy donors. Virus negative strand was detected by a Tth enzyme-based strand-specific assay and virus sequences were analysed by single strand conformation polymorphism (SSCP) and sequencing. Concentrations of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12p70 were measured in culture supernatants and respective mRNAs were analysed in cell extracts by quantitative RT-PCR. For 15 sera, HCV RNA was detectable in 2- and 3-week cultures from at least one donor. Virus negative strand was detected in 29 % of macrophage samples in this group. In four cases, HCV RNA sequences amplified from macrophages differed from those amplified from sera suggesting evolution during infection. Concentrations of TNF-alpha and IL-8 were found to be significantly higher in supernatants from HCV-infected cultures. In conclusion, these preliminary data suggest that primary human macrophages are susceptible to HCV infection in vitro and this infection is associated with the induction of cytokines TNF-alpha and IL-8.