Editorial

-

Rezistencija HIV1 virusa na 3 klase lijekova: prvi slučaj

Resistance of Human Immunodeficiency Virus (HIV) to antiretroviral drugs is an important limitation in achieving complete suppression of viral replication and therefore represents an important clinical issue. It refers especially to therapy-naive individuals infected with resistant HIV strains, e.g. individuals with transmitted drug resistance (TDR). Transmitted drug resistance mutations (TDRMs) are clinically relevant and may reduce the efficacy of antiretroviral therapy. In this paper, we report the first case of HIV-1 transmitted triple-class drug resistance in Croatia. The aim of this study was to characterize drug resistance patterns and TDRMs in the newly diagnosed, treatment-naive HIV-1 patient with such a complex resistance pattern. Sanger sequencing (SS) of the sample showed four reverse transcriptase inhibitor (RTI) resistance mutations (E44D, T215E, K103N, L100I) affecting two drug classes and two protease inhibitor resistance mutations (V32I, I47V). To characterize HIV-1 minority drug resistance variants below the detection limit of SS, deep sequencing (DS) analysis was performed. DS analysis identified the same triple class resis- tance pattern that was identified by SS with addition of several other RTI mutations. The patient described in this report is the first patient with HIV-1 triple-class resistance in Croatia and further studies will be directed toward analysing possible local onward transmission of this resistant virus.Rezistencija virusa humane imunodeficijencije (HIV) na antiretrovirusne lijekove sprječava supresiju virusne replikacije te predstavlja značajan izazov u kliničkoj medicini. Posebno valja istaknuti problem primarne rezistencije (engl. transmitted drug resistance, TDR) koja se odnosi na prethodno neliječene osobe koje su zaražene rezistentnim sojevima HIV-a. Mutacije koje su pov- ezane s primarnom rezistencijom (engl. transmitted drug resistant mutations, TDRM) su klinički značajne i mogu nepovoljno djelovati na učinkovitost antiretrovirusnog liječenja. U ovom je radu opisana prva osoba s primarnom rezistencijom HIV-a na 3 klase antiretrovirusnih lijekova u Hr- vatskoj. Cilj ovog istraživanja bio je analizirati obrasce primarne rezistencije i TDRM u novodi- jagnosticiranog i neliječenog HIV-om zaraženog pojedinca. Primjenom Sangerovog sekvenciranja (SS) dokazali smo četiri mutacije povezane s rezistencijom na inhibitore reverzne transkriptaze (E44D, T215E, K103N, L100I) koje smanjuju osjetljivost na dvije klase lijekova (nukleozidne analoge inhibitore reverzne transkriptaze i nenukleozidne inhibitore reverzne transkriptaze) kao i dvije mutacije (V32I, I47V) povezane s rezistencijom na inhibitore proteaze. U svrhu identifikacije mogućeg postojanja manjinskih rezistentnih varijante ispod granice detekcije SS-a, provedena je analiza dubinskim sekvenciranjem (DS). DS analiza identificirala je isti obrazac rezistencije na 3 klase antiretrovirusnih lijekova identificiran s SS uz nekoliko dodatnih mutacija. U ovom je radu opisan prvi slučaj primarne rezistencije HIV-a na 3 klase antiretrovirusnih lijekova, a buduća istraživanja analizirat će moguće putove transmisije ovog rezistentnog virusa u Hrvatskoj

Molecular analysis of human papillomaviruses: a clinical significance

Humani papilomavirusi (HPV) su heterogena skupina virusa koja se tijekom evolucije izvrsno prilagodila replikaciji u stanicama epitela. Perzistentna infekcija humanim papilomavirusima obvezan je etiološki kofaktor u nastanku benignih i malignih novotvorina pločastog epitela. Prepoznavanje različitosti onokogenog potencijala pojedinih genotipova HPV-a potaknulo je brojna istraživanja kliničke značajnosti molekularne heterogenosti ove skupine virusa. Molekularna analiza HPV-a značajna je ne samo u temeljnim virusološkim istraživanjima već i u humanoj medicini s posebnim naglaskom na problematiku povezanosti HPV-a s određenim tipovima tumora, razvoj klinički vrijednih dijagnostičkih testova, procjenu rizika od nastanka malignih lezija, sastavljanje kliničkih postupnika za probir, praćenje i liječenje zaraženih, razvoj cjepiva, analizu "pre-cjepne" distribucije genotipova, procjenu obuhvatnosti i učinkovitosti cjepiva i praćenje distribucije genotipova nakon uvođenja univerzalnog cijepljenja.Human papillomaviruses (HPV) are a heterogeneous group of viruses that have throughout their evolution adapted to replication in epithelial cells. Persistent infection with human papillomaviruses represents a necessary co-factor for the development of benign and malignant epithelial neoplasia. Different oncogenic potential of individual HPV genotypes encouraged numerous studies on clinical significance of the observed molecular heterogeneity of HPV. Molecular analysis of HPV is relevant not only for basic virological research but for human medicine as well with particular emphasis on association between HPV and various types of human tumors, development of clinically validated diagnostic assays, assessment of risk for progression of cervical lesions, vaccine design and development, analysis of pre-vaccination distribution of HPV genotypes, analysis of vaccine coverage and efficacy as well as monitoring of possible genotype replacement following introduction of universal HPV vaccination

Molecular Diagnostics of Sexually Transmitted Infections

Metode molekularne dijagnostike dio su rutinskoga dijagnostičkog algoritma spolno prenosivih infekcija. Molekularna dijagnostika spolno prenosivih infekcija u kliničkim laboratorijima uglavnom se temelji na primjeni standardiziranih amplifi kacijskih molekularnih testova. Najčešće se rabe metode lančane reakcije polimerazom (polymerase chain reaction, PCR), PCR u realnom vremenu (real-time PCR), tekućinska hibridizacija (hybrid capture), amplifi kacija posredovana transkripcijom (transcription-mediated amplifi cation) i tehnologija izmjene lanaca (strand displacement technology). Primjenom ovih metoda možemo detektirati uzročnike spolno prenosivih infekcija u različitim biološkim uzorcima, tj. obriscima cerviksa, vagine, uretre i u urinu.Molecular methods are a part of the routine diagnostic algorithm for sexually transmitted infections. Molecular diagnosis of sexually transmitted infections in routine clinical laboratories is based on standardized amplifi cation assays. The majority of assays are based on polymerase chain reactions (PCR), real-time PCR, soluble hybridization, transcription- -mediated amplifi cation, and strand displacement technology. Molecular methods enable us to test a variety of biological samples including cervical, vaginal, and urethral swabs, as well as urine

RECENT DEVELOPMENTS IN SEROLOGIC AND MOLECULAR DIAGNOSIS OF HEPATITIS B AND C

Opisali smo glavne novosti u dijagnostici hepatitisa B i C kao dio hrvatskih smjernica za dijagnostiku i liječenje virusnih hepatitisa 2013. Dijagnostika akutnog i kroničnog hepatitisa B započinje određivanjem HBsAg, anti-HBc i anti-HBs. Ostale serološke biljege hepatitisa B treba određivati tek u drugom koraku ako su nalazi HBsAg i/ili anti-HBc pozitivni. Pozitivan nalaz samo na anti-HBc potrebno je obvezno nadopuniti određivanjem HBV DNK. Kvantitativno određivanje HBsAg treba se koristiti komplementarno s određivanjem HBV DNK za: (i) razlikovanje inaktivnog nositelja HBsAg od aktivnog kroničnog HBeAg-negativnog hepatitisa B u bolesnika sa HBV DNK nižom od 2.000 IU/mL te za (ii) praćenje tijeka liječenja kroničnog hepatitisa B s pegiliranim interferonom-alfa. Metoda PCR-a u stvarnom vremenu ostaje i dalje metoda izbora za detekciju i kvantifikaciju HBV DNK. Testiranje za HCV započinje određivanjem specifičnih protutijela probirnim enzimskim imunotestovima ili brzim testovima. Sve osobe s pozitivnim probirnim anti-HCV testom treba testirati na prisutnost HCV RNK ili antigena virusne kapside. Potvrdne anti-HCV testove treba koristiti samo kao dodatne testove koji će potvrditi ili isključiti značenje reaktivnih rezultata probirnih enzimskih imunotestova u osoba koje su HCV RNK negativne. U praćenju virusne kinetike tijekom trojne terapije hepatitisa C preporučuje se korištenje molekularnih testova s identičnom donjom granicom detekcije (LLOD) te donjom granicom kvantifikacije. Određivanje rezistencije HCV-a na inhibitore proteaze nije dio obveznog dijagnostičkog praćenja bolesnika liječenih s trojnom terapijom. Ne postoje dostatni dokazi o potrebi uvođenja subtipizacije HCV-a u obveznu predterapijsku obradu bolesnika s kroničnim hepatitisom C. Genotip IL-28 je važan prediktor SVR-a u bolesnika liječenih kombinacijom IFN i ribavirina kao i u bolesnika zaraženih genotipom 1 liječenih trojnom terapijom. Genotipizacija IL-28B preporučuje se u predterapijskoj obradi bolesnika s kroničnim hepatitisom C. Posebno je značajan dijagnostički parametar u terapijski-naivnih bolesnika s kroničnim hepatitisom C prilikom dvojbe dvojna vs. trojna terapija.The 2013 Update of the Croatian Guidelines for the Diagnosis and Treatment of Viral Hepatitis summarizes recent developments in the diagnosis of hepatitis B and C. Determination of HBsAg, anti-HBc and anti-HBs is the initial step in the diagnostic workup of acute and chronic hepatitis B. Other hepatitis B serologic markers should be analyzed in the second stage of the diagnostic workup in HBsAg and/or anti-HBc positive patients. A positive anti-HBc finding should be followed by HBV DNA quantification. HBsAg quantification is complimentary to the HBV DNA quantification and is used: (i) to differentiate between inactive HBsAg carriers and active chronic HBeAg-negative hepatitis B in patients with HBV DNA <2000 IU/mL; and (ii) for treatment monitoring in patients with chronic hepatitis B receiving pegylated interferon-alpha. Real-time PCR remains the method of choice for detection and quantification of HBV DNA. The first step in HCV testing is determination of specific antibodies via screening assays, enzyme immunoassays or point-of-care assays. All persons with positive results of anti-HCV screening assays should be additionally tested for HCV RNA or presence of HCV viral capsid antigen. Confirmatory anti-HCV assays should be used as additional assays for confirmation of reactive results obtained by screening enzyme immunoassays in HCV RNA-negative persons only. Molecular assays with identical lower limit of detection (LLOD) and lower limit of quantification are recommended for monitoring of viral kinetics during chronic hepatitis C triple therapy. HCV resistance testing to protease inhibitors is not part of the recommended diagnostic monitoring of patients receiving triple therapy. HCV subtyping is currently not recommended as part of pretreatment diagnostic algorithm due to currently insufficient evidence on its clinical usefulness. IL-28 genotype is an important predictor of SVR in patients treated with a combination of interferon-alpha and ribavirin as well as in patients with HCV genotype 1 receiving triple therapy. IL-28B genotyping is recommended as part of pretreatment diagnostic workup in patients with chronic hepatitis C and is a particularly important parameter for recommending double versus triple therapy in treatment-naïve patients with chronic hepatitis C

Pre-vaccination prevalence of high-risk human papillomaviruses (HPV) in women from Kosovo and their related sociodemographic characteristics

Objectives: Kosovo’s current health care system does not support organized nationwide cervical cancer screening and human papillomavirus (HPV) vaccination programs. To date, no reliable data are available on cervical cancer incidence and mortality in Kosovo, or on high-risk HPV (HR-HPV) prevalence and HPV type distribution. Our aim is to determinate the pre-vaccination prevalence and distribution of HR-HPVs and to assesses the associations between sociodemographic characteristics and increased risk of HPV infection in women from Kosovo. Material and methods: Detection of HR-HPV DNA in cytologically evaluated cervical smears was performed using a clinically validated Abbott RealTime High Risk HPV test, Roche Linear Array HPV Genotyping Test, HPV52 type-specific real-time PCR and an in-house GP5+/GP6+/68 PCR. Results: The crude overall prevalence of any of the HR-HPVs was estimated at 13.1% (26/199; 95% confidence interval (CI): 9.1–18.5%), with HPV16 being the most common type (7/26, 26.9%), followed by HPV31 and HPV51, each detected in 4/26 (15.4%) cervical specimens, HPV18, detected in 3/26 (11.5%) specimens, HPV52 and HPV66, each detected in 2/26 (7.7%) specimens, and HPV33, HPV45, HPV56, and HPV58, each detected in a single (3.9%) specimen. Women over 40 (OR = 0.36), older than 18 at sexual debut (odds ratio (OR) = 0.28), those that had delivered at least one child (OR = 0.32), and those that had a history of pregnancy termination (OR = 0.39) were at lower risk for HPV infection. Conclusion: Because more than 70% of cervical precancerous lesions could have been prevented in Kosovo using nationwide HPV-based cervical cancer screening and HPV vaccination, it is of outmost importance to implement both programs in the national health care system as soon as possible

Imported Case of Hepatitis A from Burkina Faso - a Case Report

U radu opisujemo mladu bolesnicu sa šećernom bolesti tipa I koja se zarazila virusom hepatitisa A (HAV) genotipa IB tijekom boravka u Burkini Faso. Prikaz bolesnice nedvojbeno potvrđuje važnost pridržavanja preporuka stručnih društava i nadležnih tijela o cijepljenju putnika.We describe a clinical case of hepatitis A in a young women with type I diabetes infected with genotype IB of hepatitis A virus (HAV) from Burkina Faso. The case confirms the importance of strict adherence to the recommendations by professional associations and institutions regarding vaccination of the travellers

Treatment of chronic hepatitis C in Croatian war veterans: experiences from Croatian reference center for viral hepatitis

Aim. To examine the risk factors, comorbidity, severity of liver disease, treatment course, and outcome in Croatian war veterans with chronic hepatitis C, especially those suffering from posttraumatic stress disorder (PTSD). ----- Methods. We collected medical records of 170 adult men diagnosed with chronic hepatitis C who started treatment with a combination of pegylated interferon-alpha and ribavirin between January 2003 and June 2009 at the Croatian Reference Centre for Viral Hepatitis. ----- Results. Participants' mean age was 43±9 years. Among 170 participants, there were 37 war veterans (22%). The main risk factor in veteran patients were operative procedures with transfusions (46% vs 5% in non-veterans; P<0.001) and in non-veteran patients intravenous drug use (42.1% vs 13%; P<0.001). The average duration of infection was longer in war veterans (14.5±3.4 vs 12.2±7.2 years; P=0.020). The percentage of PTSD comorbidity in the whole group was 11% (18/170) and in the war veterans group 49% (18/37). The prevalence of sustained virological response in patients with PTSD was 50% and in patients without PTSD 56%. Treatment reduction in patients with PTSD (33%) was higher than in patients without PTSD (12%; P=0.030). ----- Conclusion. Croatian war veterans are a group with high risk of chronic hepatitis C infection because many of them were wounded during the Croatian War 1991-1995. Considerations about PTSD as a contraindication for interferon treatment are unjustified. If treated, patients with PTSD have an equal chance of achieving sustained virological response as patients without PTSD

The Need for Systematic Monitoring and Improved Surveillance of Hepatitis C Patients in Croatia

Aim: The aim of this study was emphasizing the need for a more systematic monitoring of patients diagnosed with HCV in Croatia. Methods: From 2014 to 2018, at the University Hospital for Infectious Diseases, sera from 23,524 patients were tested for HCV. Confirmatory testing was performed by Western Blot. Adult patients with an anti-HCV positive screening test were analysed. HCV RNA was quantified by real-time PCR, while HCV genotypes and subtypes were determined by PCR and the reverse hybridization method. Results: A total of 428 anti-HCV ELISA-positive adults were analysed (68.7% males, 31.3% females, median age 43 years, range 19-88 years). Hepatitis C was confirmed by WB in 390, while 28 patients had borderline WB results. Anti-HCV was not confirmed by WB in 10 patients. HCV RNA was tested in 331 patients and viremia was detected in 218 patients. There was no data on HCV RNA in 97 patients (22.66%). HCV genotypes/subtypes were determined in 185 of 218 anti-HCV WB positive patients. Genotype 1 was detected in 97/185 (52.43%), genotype 2 was detected in 3/185 (1.62%), while subtype 3a was detected in 76/185 (41.08%) and genotype 4 in 9/185 patients (4.86%). Conclusion: In a five-year period, the HCV seroprevalence rate in subjects tested at the University Hospital for Infectious Diseases was 1.81%. According to the data analysed, almost one quarter of patients with detected anti-HCV antibodies were not treated further, which indicates the need for a systematic monitoring of patients diagnosed with HCV. It is necessary to determine viremia after a positive anti-HCV screening result in order to initiate treatment and prevent HCV-related complications. (Radmanić L, Cetinić Balent N, Šimičić P, Vince A, Židovec Lepej S, Đaković Rode O. The Need for Systematic Monitoring and Improved Surveillance of Hepatitis C Patients in Croatia. SEEMEDJ 2020; 4(2); 28-34