Mirasol PRT system inactivation efficacy evaluated in platelet concentrates by bacteria-contamination model

Background/Aim. Bacterial contamination of blood components, primarily platelet concentrates (PCs), has been identified as one of the most frequent infectious complications in transfusion practice. PC units have a high risk for bacterial growth/multiplication due to their storage at ambient temperature (20 ± 2°C). Consequences of blood contamination could be effectively prevented or reduced by pathogen inactivation systems. The aim of this study was to determine the Mirasol pathogen reduction technology (PRT) system efficacy in PCs using an artificial bacteria-contamination model. Methods. According to the ABO blood groups, PC units (n = 216) were pooled into 54 pools (PC-Ps). PC-Ps were divided into three equal groups, with 18 units in each, designed for an artificial bacteria-contamination. Briefly, PC-Ps were contaminated by Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli in concentrations 102 to 107 colony forming units (CFU) per unit. Afterward, PC-Ps were underwent to inactivation by Mirasol PRT system, using UV (l = 265-370 nm) activated riboflavin (RB). All PC-Ps were assayed by BacT/Alert Microbial Detection System for CFU quantification before and after the Mirasol treatment. Samples from non-inactivated PC-P units were tested after preparation and immediately following bacterial contamination. Samples from Mirasol treated units were quantified for CFUs one hour, 3 days and 5 days after inactivation. Results. A complete inactivation of all bacteria species was obtained at CFU concentrations of 102 and 103 per PC-P unit through storage/ investigation period. The most effective inactivation (105 CFU per PC-P unit) was obtained in Escherichia coli setting. Contrary, inactivation of all the three tested bacteria species was unworkable in concentrations of ≥ 106 CFU per PC-P unit. Conclusion. Efficient inactivation of investigated bacteria types with a significant CFU depletion in PC-P units was obtained - 3 Log for all three tested species, and 5 Log for Escherichia coli. The safety of blood component therapy, primarily the clinical use of PCs can be improved using the Mirasol PRT system

Principles of NAT technology in view of the results obtained at the Institute of Transfusiology of MMA from 2007 till 2011

Nucleic-acid Amplification Testing (NAT) consist of extraction, amplification, hybridization and detection. Polymerase Chain Reaction (PCR) is method for amplification of viral genetic material (DNA/RNA) in enough number of copies, so we can detect them. Since year 1943, when transmission of hepatitis via transfusion was reported till beginning of 21st century, when NAT was implemented, large number of tests for detection of hepatitis B and C virus (HBV, HCV) and human immunodeficiency virus (HIV) were developed. In this article, we describe basic princples of detection of these viruses by NAT. We also show results from testing samples of blood donors in Institute of Transfusiology from April 2007 till June 2011. From 50 369 donors testing in Mini Pools (MP) of 24, we found two HCV RNA positive samples, one HBV DNA positive sample and all pools were HIV RNA negative. From 2 689 samples which were testing by Individual Donation (ID) NAT, 135 were HBV DNA positive, 108 were HCV RNA positive and 7 were HIV RNA positive. All samples also were tested by Enzyme Link Immunosorbent Assay (ELISA) and Confirmatory tests. Comparison of our results show validity of NAT testing, especially when ELISA tests of less specificity and sensitivity were used.Da bi testiranje nukleinskih kiselina bilo moguće, neophodno je izdvojiti ih iz virusa (ekstrakcija), zatim umnožiti (amplifikacija), obeležiti specifičnim probama (hibridizacija) i detektovati. To se obavlja korišćenjem metode 'Polymerase Chain Reaction' (PCR) ili lančane reakcije polimeraze koja nam omogućava da umnožimo deo virusnog genoma do količine koju je moguće detektovati. Od 1943. godine, kada je transmisija hepatitisa preko transfuzije prvi put prijavljena, do početka 21. veka, kada je masovno uvedena NAT tehnologija (eng. Nucleic-acid Amplification Testing - testiranje amplifikovanih nukleinskih kiselina), došlo je do značajnog poboljšanja u otkrivanju virusa uzročnika hematogenih transmisivnih bolesti. U ovom radu dati su osnovni principi NAT-a, odnosno detekcije genetskog materijala virusa uzročnika hepatitisa tipa B, C i virusa humane imunodeficijencije (HIV-a). Takođe, opisani su NAT rezultati dobijeni kod uzoraka krvi davalaca u Institutu za transfuziologiju VMA u periodu od aprila 2007. do juna 2011. godine. Od 50.369 testiranih davalaca u pulu, detektovana su dva HCV RNK pozitivna uzorka, jedan HBV DNK pozitivan uzorak, a za HIV RNK svi pulovi su bili negativni. Od 2.689 pojedinačno obavljenih NAT testiranja, najviše pozitivnih bilo je za HBV DNK (135 uzoraka), zatim za HCV RNK (108 pozitivnih) i 7 pozitivnih za HIV RNK. Za sve navedene uzorke paralelno su rađeni određeni enzimoimunski i potvrdni testovi. Komparacija rezultata primenjenih testova na našim ispitanim uzorcima potvrdila je opravdanost primene NAT-a, posebno prilikom korišćenja manje specifičnih i osetljivih enzimoimunskih testova.nul

Sociodemographic profile of opiate addicts at increased risk of infection with HBV, HCV, HIV, Тreponema pallidum, Cryptococcus neoformans, Pneumocystis carini and WNV

Uvod. Zavisnici od opijata predstavljaju visokorizičnu grupu zbog međusobnih infekcija krvno-prenosivim bolestima, vertikalne transmisije patogena, kao i zbog mogućnosti da budu potencijalni donori krvi (naročito kao plaćeni davaoci). Cilj. Cilj našeg istraživanja bio je određivanje socio-demografskog profila 99 zavisnika od opijata Šumadijskog okruga lečenih u Kliničkom centru Kragujevac supstitucionom terapijom metadonom i buprenorfinom, kao i određivanje prevalence infekcija krvno-prenosivim patogenima: virus hepatitisa tip B, virus hepatitisa tip C, virus stečene imunodeficijencije (HBV, HCV, HIV) i sifilis (Treponema pallidum), kao i Cryptococcus neoformans, Pneumocystis carini i virus Zapadnog Nila (West Nile Virus – WNV). Metode: Ispitanici su odgovarali na pitanja iz Pompidu upitnika i podaci iz ovog upitnika su korišćeni za analizu osnovnih socio-demografskih karakteristika. Svi uzorci su testirani korišćenjem ELISA (Enzyme – Linked ImmunoSorbent Assay)/CIA (Chemiluminescent Immuno – Assay) testova za virus hepatitisa tip B, virus hepatitisa tip C, virus stečene imunodeficijencije (HBV, HCV, HIV) i sifilis (Treponema pallidum), kao i korišćenjem PCR (Polymerase Chain Reaction) za HBV, HCV, HIV, Cryptococcus neoformans, Pneumocystis carini i virus Zapadnog Nila (West Nile Virus – WNV). Rezultati: Najveći broj ispitanika je bio muškog pola (81,8 %), starosti 32 (19 – 57) godine, 99 % je živelo u gradu, nezaposlenih je bilo 58,6 %, sa završenom srednjom školom 67,7 %, a korisnika neadekvatne primene igala 34,3 %. Netestiranih na HBV je 39,4 %, na HCV 36,4 %, HIV 28,3 %, a samo njih 4 (4 %) je primilo vakcinu protiv HBV. Što se tiče analiza na prisustvo HBV infekcije, ELISA/CIA i PCR negativnih je bilo 66, HBV ELISA/CIA i PCR pozitivnih je bilo 19, HBV ELISA/CIA-negativnih / PCRpozitivnih 12 i HBV ELISA/CIA-pozitivnih / PCR-negativnih 2 ispitanika. Testiranje na HCV infekciju je pokazalo sledeće: ELISA/CIA i PCR negativnih ispitanika je bilo 15, HCV ELISA/CIA i PCR pozitivnih je bilo 58, HCV ELISA/CIA-negativnih / PCRpozitivnih 11, a HCV ELISA/CIA-pozitivnih / PCR-negativnih 15. Svi ispitanici su bili negativni na HIV (ELISA/CIA i PCR testiranje), kao i na patogene oportunističkih infekcija (Cryptococcus neoformans; Pneumocystis carini; PCR testiranje) i na prisustvo WNV (PCR testiranje). Jedan ispitanik je bio pozitivan na sifilis (ELISA testiranje). Zaključak: Naši rezultati su pokazali da je pozitivnost na prisustvo patogena krvnoprenosivih bolesti HBV i HCV visoka u ispitivanoj grupi zavisnika od opijata i iznosi 33,4 % i 84,8 %. Preporuka bi bila da oni budu periodično testirani na prisustvo HBV, HCV i HIV, komplementarnim ELISA/CIA i PCR testovima, obzirom na izvestan stepen diskrepance u dobijenim rezultatima serološkog i molekulskog testiranja.Background/Aim. Intravenous drug users (IDUs) are still a high risk-group for cross-reacting blood-borne infections, for vertical pathogen transmission, as well as for potentially blood/plasma donation (particularly as "payed" donors). The aim of our study was to establish the profile of opiate addict and prevalence of blood-borne pathogens – Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human Immunodeficiency Virus (HIV), Treponema pallidum, Cryptococcus neoformans, Pneumocystis carini and West Nile Virus – WNV among 99 patients on substitution therapy with methadone and buprenorphine from Shumadia District. Methods. The Treatment Demand Indicator (TDI) of Pompidou-questionnaire was used to assess the history of drug abuse and risk behavior. All blood samples were tested for HBV, HCV, HIV and Treponema pallidum by Enzyme-Linked ImmunoSorbent Assay (ELISA) or Chemiluminescent Immuno-Assay (CIA). Investigations were also performed for HBV, HCV, HIV, Cryptococcus neoformans, Pneumocystis carini and West Nile Virus – WNV by molecular testing – Polymerase Chain Reaction (PCR) method. Results. The majority of patients were male (81.8 %), median age 32 (19 – 57) years, lived in a city (99 %), unemployed (58.6 %), with finished secondary school (67.7 %), unsafe injecting practices (34.3 %) and never previously tested for HBV (39.4 %), HCV (36.4 %) nor HIV (28.3 %); only four percentage of them previously got HBV-vaccine. Complementary testings resulted with the following results: HBV ELISA/CIA and PCR negativity for 66 patients and positive results (by ELISA/CIA and PCR) for 19 patients. However, a difference was observed in ELISA/CIAnegative / PCR-positive result for 12 and ELISA/CIA-positive / PCR-negative for two patients, respectively. Further, negative results for HCV (ELISA/CIA and PCR testing) were found in 15 IDUs and positive results (using both methods) were found in 58 patients. Different results for ELISA/CIA-negative / PCR-positive results were found in 11 IDUs and ELISA/CIApositive / PCR-negative results were found in 15 patients. All investigated IDUs were negative for HIV (ELISA/CIA and PCR testing) and for pathogens of opportunistic infection (Cryptococcus neoformans; Pneumocystis carini; PCR testing), as well as for West Nile Virus (PCR testing). Just one IDU was positive for syphilis (ELISA and confirmatory testing). Conclusion. This study undoubtedly confirmed the effectiveness and improved safety of originally designed complementary (ELISA/CIA and PCR) pathogen monitoring system. Our study demonstrated that the positivity for HBV and HCV is still very high (33.4 % and 84.8 %, respectively) in IDUs. Thus, we suggest that drug users have to be periodically screened using a complementary serological/molecular testing, also concerning differences/discrepancies in results obtained using these methods