Hepatitis B and blood transfusion

Hepatitis B is caused by infection with pandemic hepatitis B virus (HBV) and has a serious impact on blood safety worldwide. Active immunization is an efficient preventive measure particularly in countries with medium and high HBV prevalence. However, as long as only some countries have started immunization programmes for their younger population and escape mutants occur, stringent donor selection and testing is presently the only approach to reduce the risk of transfusion transmitted HBV infections. Surrogate testing for alanine aminotransferase (ALT) has shown its limitations and has mostly been discontinued especially since sensitivity and specificity of hepatitis B surface antigen (HBsAg) tests have been continuously improved. However, there remains a significant risk by donations from freshly infected donors who have not yet developed detectable HBsAg levels and donations by donors with occult infection that are HBsAg‐negative but HBV DNA‐positive. Nucleic acids amplification technology (NAT) has been applied primarily to close the diagnostic window between HBV infection and HBsAg detection and has shown to be effective either in testing donor samples in mini‐pools or by individual donation (ID) testing. It has also been shown that mini‐pool NAT may not be sufficiently sensitive to identify occult chronic carriers who show very low virus concentrations in their plasma. For those chronically infected donors, highly sensitive mini‐pool NAT with specific enrichment procedures and ID NAT may be required. Anti‐HBc testing would be the most sensitive approach resulting in highest yield rates for occult carriers. However, for medium and high prevalence countries, deferral rates would be extremely high with negative effects on the blood supply. Inactivation would be a reasonable alternative but at very high costs and is presently not applicable to all blood components. Future scenarios will most probably be diverse, depending on the prevalence in a given country, its resources and the vaccination status of its population.     

High prevalence of HIV p24 antigen among HIV antibody negative prospective blood donors in Ile-Ife,Nigeria

Blood transfusion service centers in Nigeria screen donated blood for markers of HIV infection using antibody- (Ab) based rapid test and in some centers, positives are re-tested using Ab-based ELISA. Paucity of data exists on p24 antigen prevalence among HIV Ab-negative donors in Nigeria. This study aims at detecting HIV p24 antigen among prospective blood donors in Osun State, Nigeria. Prospective blood donors negative for HIV antibodies using Determine test kit were re-tested using BIORAD GENSCREEN Ultra Ag-Ab ELISA kit, a fourth-generation ELISA kit that detects HIV antibodies/p24 antigen. Of the 169 HIV Ab-negative prospective donors, 10 (5.9%) were positive for HIV p24 antigen and 70% (7/10) of them were in the age range 18–30 years. Results of this study show that blood transfusion is still one of the major routes of HIV transmission in Nigeria and a higher proportion is among youth. Inclusion of p24 antigen testing into the blood donor screening will help reduce transfusion associated HIV in Nigeria if Nucleic Acid Testing (NAT) of all blood donor samples is not affordable; also, HIV enlightenment programs tailored toward youth may help reduce this rate among donors since more young people donate blood in low/middle-income countries than in high-income countries.     

Epidemiological surveillance of blood donors and residual risk of blood-borne infections in France, 2001 to 2003]

The national surveillance of French blood donors is performed by the Institut de Veille Sanitaire and the National Reference Center for Hepatitis B and C in transfusion in collaboration with the Etablissement Fran?ais du Sang and the Army blood center. The main objectives of this surveillance are to evaluate trends in prevalence and incidence rates of blood-borne infections in the blood donor population, to identify routes of contamination and to assess residual risk. This exhaustive surveillance also contributes to evaluate the blood donor selection and the impact of measures taken to prevent infections in the general population. The analyse of the database of all blood donations obtained from 2001 to 2003 has shown that prevalence rates were stable in the study period (0.60 per 10(4) donors for HIV, 8.0 per 10(4) donors for HCV, 1.8 per 10(4) first-time donors for HBs Ag and 0.56 per 10(4) donors for HTLV), The incidence rate of HIV and HBV (1 per 10(5) person-years) was three-times higher than for HCV (0.35 per 10(5) person-years) and eleven times higher than for HTLV (0.09 per 10(5) person-years). At least, the residual risk of transfusion-transmitted viral infections is very low: 1/3,150,000 donations for HIV, 1/10,000,000 donations for HCV and 1/640,000 donations for HBV. The yield of Nucleic Acid Testing (NAT) is limited since only 2 donations for HIV and 3 for HCV which were negative for antibodies were discarded thank to the NAT.     

TTID risk in the Latin America and Caribbean: recommendations to improve safety

Transfusion-Transmitted Diseases (TTD) have been a persistent risk since the onset of transfusion medicine. In Latin America and the Caribbean countries (LACC), prevention of TTD requires special and different care in comparison with the in United States and European countries, due to the still persistent high prevalence of replacement donors, specific geographic location, topography, and climate, genetic and cultural and socio-economic status of the population. These factors result in different distribution patterns of the infections, which together with the migration have changed the donor profile. According to the Pan American Health Organization (PAHO) reports, the screening of HIV, HBV, HCV and T. pallidum has increased in LACC since 2005, significantly reducing the risk of transmission. However these results have not yet reached the established aim in 1998, of analyzing 100% of blood units for these pathogens. The strategies recommended for the LACC should be based on the following points: a) the establishment of national blood transfusion services, b) blood collection from volunteer donors, c) high quality monitoring of TTD and emerging transfusion-transmitted infections (ETTI) in all blood units, d) implementation of effective quality assurance systems and e) the rational use of blood components and alternatives to transfusion.     

Testing for established viruses: from the screening to the confirmation

Enzyme Immunoassays (EIAs) detecting antibodies (Ab) or antigen (Ag) used to avoid viral transmissions by transfusion, are the most suitable methods for blood screening because they are economical and adaptable to high-throughput testing. The continuing need to improve screening for HIV and HCV, led to develop Ag/Ab combination assays. The benefit of such assays has been widely demonstrated, since they are able to detect infected individuals in viremic pre-seroconversion phase, making them particularly attractive for ensuring blood safety when nucleic acid testing (NAT) cannot be implemented. In resource-limited countries, especially in remote-areas, blood safety is often based upon rapid tests (RT) which are easy to handle and can be used with whole blood. Nevertheless, the poor sensitivity of RT compared to EIAs could significantly impair blood safety. Because NAT could efficiently detect serologically negative donors, many developed countries have implemented this method in blood screening. NAT yields vary depending on epidemiological situations. NAT is subjected to false-negative results due to viral diversity or to extremely low viral loads, but also to false-positive due to sample cross-contamination. A repeatedly NAT reactive sample with multiplexed NAT is submitted to a virus-specific amplification testing to discriminate between the viruses, creating some problematic situations consisting of samples reactive with the screening assay but non-reactive with the discriminatory assay. Although the primary purpose of viral screening testing is to prevent pathogen transmission to recipients, confirmatory testing intends to manage donors with adverse test results and to guarantee an optimal blood supply. As EIAs show a low positive predictive value in low-prevalence countries due to frequent non-specific reactions, the use of confirmatory assays is justified. Nevertheless confirmatory tests are known to be less sensitive than EIAs in the early phase of infection and show also nonspecific reactivity leading to indeterminate results which create difficulties in managing donors. As in limited-resource countries, specific confirmatory assay cannot be used, alternate strategies for confirmatory testing have been proposed, as to simultaneously test blood with two assays and to exclude donors reactive with both, or to use screening test signal values to support confirmation. Although the direct detection of the virus through its genome might be considered as the best solution to ensure blood safety, the choice of blood screening strategy should be adapted to local epidemiology and organization constraints.     

Profile of transfusion transmissible infections and associated risk factors among blood donors of Kerala

Blood transfusion is an important mode of transmission of infections to the recipient. The aim of the study was to assess the prevalence of transfusion transmissible infections and the possible risk factors among blood donors. During the study period (1994-99) donor samples were screened for HBsAg, HIV, HCV and Syphilis. The relevant donor history was examined to identify the risk factors leading to transfusion transmissible infections. The prevalence of infection was 3.1% among donors, with HBsAg constituting 1.3%, HCV 1.4%, HIV 0.2% and RPR 0.2%. In a classification based on occupation, class III donors (drivers, Businessmen & men in uniform) showed a significantly higher incidence of transfusion transmissible infections for HIV (0.38%) and Syphilis (0.36%). Health care workers constituted only a small fraction of the study; prevalence of infections was low among them. History of jaundice or hospitalization was not associated with higher incidence of seropositivity. Seropositivity for HIV is relatively low compared to similar studies conducted in other regions of the country. This finding is attributed to the pre donation counseling in donor selection. An important outcome of the study is that Class III donors form a high-risk group for transfusion transmissible infections.     

Surveillance of the genetic variation in incident HIV, HCV, and HBV infections in blood and plasma donors: implications for blood safety, diagnostics, treatment, and molecular epidemiology

Surveillance for molecular variants in blood donors is vital to assuring that blood screening and supplemental assays are sensitive to circulating strains of blood-borne viruses. Blood screening and diagnostic assays licensed in the United States are largely based on prototype viral strains. Documentation of divergent viral strains in the donor pool can lead to accelerated development and licensure of robust serologic and nucleic acid amplification (NAT) assays for donor screening and diagnostic applications. In addition, surveillance for viral variants among donors has implications for assessing the prevalence of drug and vaccine escape mutants and for detecting and monitoring rare variants that may be newly introduced or increasing in the United States donor population. Combined NAT and serologic screening, supplemented by novel serologic testing strategies, can be used to identify donors with incident infections, which are of particular interest with respect to blood safety and public health implications. A systematic program is proposed for the genetic characterization of viral genomes in donors with incident HIV, HCV, or HBV infections.     

Virus de l’hépatite E,implications en transfusion sanguine

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.     

Chagas disease: a threat to safe blood transfusion

Chagas disease affects approximately 8 million infected people in Mexico and Central and South America and causes 12 500 deaths annually; 41 200 new cases are reported annually (2006). Coordinated multicenter programmes have decreased about 70% of new infections in South America due to the interruption of vectorial and decreased blood transfusion transmissions. Migration of the infected population from rural areas to urban centres in endemic and non-endemic countries led to the urbanization and globalization of Chagas disease. Chagas disease is now an emerging disease in non-endemic areas, where congenital, blood and organ transplant transmissions are associated with reactivation of chronic Chagas disease in patients under immunosuppression or HIV infection. Recent initiatives in non-endemic countries have been implemented to control its transmission by blood transfusion and transplants of organs. Most of the infected people were asymptomatic individuals in the chronic phase of the disease, characterized by and low and intermittent parasitemia and diagnosed by serological tests. In endemic countries, universal mandatory screening tests for detection of Trypanosoma cruzi antibodies dramatically reduced blood transfusion transmission. In non-endemic countries, where transfusion represents the most important way of transmission, different strategies have been registered: (1) blood donor selection and deferral; (2) blood donation testing; (3) blood donation testing in people who lived in endemic areas. Questionnaires containing well-defined questions help to identify patients from endemic areas and their epidemiological data but are of limited value since they depend on donor information. High-performance serologic tests were reported for the detection of anti-T. cruzi antibodies like ELISA with epimastigotes or trypomastigotes or recombinant antigens, chemiluminescent, immunoblot and radioimmunoprecipitation assays. Almost 100% of sensitivity and specificity were registered when tested in samples of well-defined chronic chagasic patients but gold standards are not validated in circumstances of low prevalence of the disease. Additionally, their value in cases of inconclusive results (one positive test and other negative or doubtful results) was not known. Unfortunately, molecular methods are less sensitive than serology for the chronic phase of the disease (45–100% sensitivity) and were not reliable for screening tests in blood banks. Perspectives of the reduction of parasites by photochemical treatment and ultraviolet (crystal violet, methylene blue, amotosalen, riboflavin, thiopirilium) were reported sometimes with slight changes in the blood products. However, the main challenge is to demonstrate their value for prevention of a broad spectrum of agents transmitted by blood transfusion (including príon disease) and inactivation of low parasite load. In summary, questionnaires including specific questions about donor epidemiology and high-performance serologic tests have been useful for blood bank screening. The cost–benefit–effectiveness analyses of universal serologic screening vs. serologic screening of selected donors depend on the prevalence of positive donors and should be evaluated in different regions. Finally, as pathogen inactivation methods may represent remarkable improvement in blood bank transfusion, efforts from the academia are necessary to prove their safety and effectiveness, and from blood banking–transfusion medicine community and public regulators to their implementation aiming to increase the safety of blood transfusion for patients around the world.     

The detection of hepatitis C virus in South Hungary.

BACKGROUND: More than 100 million people are infected with hepatitis C virus (HCV) worldwide. The prevalence of HCV infection varies from country to country and the natural history of hepatitis C infection is not well understood. OBJECTIVES: The prevalence of anti-HCV positive blood donors in South Hungary was determined. Potential risk factors of HCV transmission were investigated and compared to anti-HCV-negative blood donors. Furthermore, the rate of anti-HCV positivity in children who had received one or more blood transfusions prior to the implementation of anti-HCV blood donor screening was evaluated. STUDY DESIGN: A total of 45719 blood donors and 120 children were tested for the presence of anti-HCV antibodies by second- and third-generation enzyme immunoassays. Positive results were confirmed by a recombinant immunoblot assay. Data on potential sources of HCV transmission were obtained by interviews. RESULTS: Among blood donors, the rate of confirmed HCV antibody-positives was 0.4% (195 of 45719 donors). Previous surgery, transfusion, more than three pregnancies, and tattoos were significantly correlated with confirmed anti-HCV positivity. Two of 120 children (1.7%) were confirmed anti-HCV positives. In both of them, serum HCV RNA could be detected. CONCLUSIONS: The prevalence of anti-HCV positive blood donors in South Hungary is low. Nosocomial infections and tattooing were found to be the most important risk factors for transmission of HCV. Because of the low prevalence of anti-HCV positive blood donors, only a small number of children, who received blood transfusions prior to the implementation of anti-HCV blood donor screening, are infected with HCV.     

ALT单项不合格献血者再次献血血液筛查结果追踪调查分析

目的探讨和评价ALT血液筛查项目在临床用血血液安全中的意义。方法采用回顾性调查法对2010年10月至2012年12月期间,ALT单项阳性并再次献血的献血者血液筛查结果进行追踪调查和分析。结果 3910例再次献血者中,HBsAg、抗-HCV、抗-HIV、抗-TP以及ALT血液筛查结果均合格3053例,占再次献血的78.1%,ALT单项仍为阳性845例,占不合格的98.6%（845/857）,ALT阴性而ELISA筛查项目阳性9例,核酸筛查阳性3例,其中ALT合并NAT阳性1例。结论 ALT血液筛查在肝炎病毒感染＂窗口期＂或隐匿性感染中有提示的作用,在一定程度上能减少漏检的发生,在保障血液安全中具有一定的意义。     

Transfusion transmission of hepatitis B virus: still learning more about it

Background Despite continuous technical improvement in blood donation testing, hepatitis B infection remains a major risk of transfusion-transmitted viral infection. The residual risk of hepatitis B virus (HBV) transmission is related to the pre-seroconversion window period (WP), infection with immunovariant viruses, and with occult carriage of HBV infection (OBI). Results and discussion Reduction of HBV residual risk is achieved by developing more sensitive HBsAg test, by adopting anti-HBc screening when appropriate, and recently by implementing HBV nucleic acid testing (NAT), either in minipools or more efficiently in individual donations. Compared with serological testing, HBV NAT combines the ability to significantly reduce the window period and to detect OBIs. Clinical observations suggest lower transmission rate of occult HBV than WP. Lower transmission rate might be related to the low viral load generally observed in OBI donors or to the presence of defective variants associated with occult carriage. In addition, there is evidence that OBI-infected donors carrying neutralizing anti-HBs (∼50%) are unlikely to be infectious, while those with anti-HBc only may be more infectious especially in immunocompromised recipients. Immunodeficient elderly and patients receiving immunosuppressive treatments (organ transplantation or cancer chemotherapy) may be susceptible to infection with lower infectious dose even in the presence of anti-HBs. There is no evidence that blood from anti-HBc only positive/HBV DNA-negative donors is infectious. Present evidence suggests that the association of HBsAg and sensitive NAT screening adequately covers risks of HBV transfusion–transmission, allowing to dispense or discontinue anti-HBc screening, avoiding discarding non-infectious and precious blood units, particularly in high prevalence areas. Identification of ‘post-transfusion’ HBV infection relies essentially on clinical evidence of acute infection or on look-back exercises following the identification of HBV DNA-positive donations. To protect from HBV infection the increasing number of immunodeficient recipients, maximum sensitivity of HBV NAT is required. As these recipients are also at risk of viral reactivation when previously exposed, anti-HBc testing and storage of a pre-transfusion sample when anti-HBc-positive would prevent the difficulties of differentiating between transfusion–transmission and reactivation.     

Failure and success of HIV tests for the prevention of HIV-1 transmission by blood and tissue donations

French blood banks recently implemented nucleic acid testing (NAT) of all blood donations to reduce the risk of HIV transmission during the pre-seroconversion period. For tissue donation, HIV infection screening relies on HIV p24 antigen and anti-HIV-1 and 2 antibody detection. In this report, two related cases of infectious donations are described from a cornea donor during the preseroconversion window who was infected by an HIV antibody and NAT negative blood donor. After investigation, the blood donor was found to be herself in the preseroconversion window. Two months after donation, she was found to be HIV positive. The residual risk of HIV infectious blood donations since NAT has been introduced is estimated to be lower than one out of 2.5 millions. Individual NAT instead of minipool testing would not increase significantly the blood transfusion safety. In contrast, introduction of NAT should be considered to increase tissue donation safety as soon as such screening will be possible technically.     

Organ donor screening using parallel nucleic acid testing allows assessment of transmission risk and assay results in real time

Expansion of the donor pool may lead to utilization of donors with risk factors for viral infections. Donor laboratory screening relies on serological and nucleic acid testing (NAT). The increased sensitivity of NAT in low prevalence populations may result in false-positive results (FPR) and may cause unnecessary discard of organs.We developed a screening algorithm to deal, in real time, with potential FPR. Three NAT assays: COBAS AmpliScreen assay (CAS), AmpliPrep Total Nucleic Acid Isolation/CAS, and AmpliPrep/TaqMan assays, were validated and used in parallel for prospective screening of increased-risk donors (IRD), and the probability of FPR was calculated. The lower limit of detection of this algorithm was 9.79, 21.02, and 4.31 IU/mL for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, respectively, with an average turn-around-time of 7.67 h from sample receipt to result reporting. The probability that a donor is potentially infectious with two NAT concordant results was >90%. NAT screening of 35 IRD within 18 months resulted in transplantation of 102 additional organs that without screening would either not be used or used with restrictions in Australia. Using a parallel testing algorithm, real-time confirmation of seropositive donors allows use of organs from IRD and safer expansion of the donor pool.     

Where will pathogen inactivation have the greatest impact?

Blood safety has always been a major task in transfusion medicine. A strategy to obtain this aim should include donor education, donor selection, and testing of blood donations. Pathogen inactivation adds another level of safety. In the fractionation industry, pathogen inactivation methods are mandatory. Several countries also use pathogen‐inactivated plasma – from pools or single donors. Concerning the cellular blood components, there is still no method available for red cell concentrates, whereas methods for platelet concentrates are available in some countries and others are in the pipeline for commercialization. The efficiency of the ‘old’ methods to increase blood safety and the costs of the methods seem to be major obstacles for the introduction of the systems. There are also concerns on product quality and loss of volume during the inactivation process. As the importance of pathogen inactivation is largest in countries with blood donors who carry infections it is impossible to protect against, either due to high incidence of the infection or due to shortage of tests, cost will be a major question when pathogen inactivation is considered. Pathogen inactivation of red cell concentrates will also be a necessity. When pathogen inactivation methods are available for all blood components, they will have great impact to protect the patients in countries where a high percentage of the population is infected by agents transmissible through blood transfusion, and in all situations to protect against new pathogens and ‘old’ pathogens that become more virulent. The total risk of contracting infectious diseases through blood transfusion will probably be important when implementation of new methods for pathogen inactivation is considered.     

Hepatitis C infection: recent insights relevant to transfusion safety

Hepatitis C virus (HCV) infects over 100 million persons worldwide, and is hence one of the most prevalent and clinically significant blood-borne pathogens. The introduction of HCV antibody screening in the early 1990s led to detection of large numbers of infected donors and dramatic increases in blood safety. The seroprevalence of HCV in blood donors varies, ranging from rates as low as 0·01% in South Africa to ~0·5% in most developed countries, to > 5% in focal endemic areas such as Egypt. Studies of HCV in infected donors and recipients were instrumental in the discovery of HCV, and have yielded ongoing insights into HCV epidemiology, natural history and pathogenesis. Recent examples include detailed characterization of the dynamics of acute HCV infection based on studies of plasma donor panels, and important contributions to understanding viral and host factors influencing HCV transmission and disease outcome based on linked donor-recipient cohort studies. The addition of nucleic acid testing (NAT) for HCV RNA to routine HCV antibody screening of donors in 1999 further reduced the risk of transfusion-transmitted HCV. Mini-pool (MP)-NAT screening has led to detection of well over 500 donors in the viraemic, preseroconversion phase of primary HCV infection, as well as to discrimination of seropositive donors into those with resolved and chronic infections. Studies of donors with acute, chronic/persistent and resolved HCV infections have further contributed to our understanding of viral and host genetic and immunological determinants of spontaneous clearance of HCV and short- and long-term disease outcomes. This review will highlight these past scientific contributions, and present the results of a recent survey of the yield of HCV MP-NAT screening and estimated residual risks of MP-NAT screened transfusions in different regions of the world. Finally, the review will discuss available evidence for the infectivity of very low-level viraemia that may be missed by MP-NAT, information that is pivotal to modelling the residual risk of MP-NAT screened blood transfusions, and which will guide further strategies to reduce risk by further enhancing screening or implementation of pathogen-inactivation technologies.     

Current concepts in serological testing – TTID

Introduction Blood components play a major part in a huge number of therapeutic interventions in oncology, haematology, surgery and other medicine disciplines. Therefore, the supply of safe blood products is a major ongoing challenge for blood transfusion services. Serology screening tests were developed to improve blood safety. As screening for antibodies reflects an immune reaction in response to infection, the diagnostic window period is longer for antibody tests than for direct screening assays, such as nucleic amplification tests (NAT). The introduction of mini-pool NAT (MP-NAT) and individual donation NAT (ID-NAT) as well as the development of antigen screening assays, such as the hepatitis C virus antigen test (which detects the NS3 antigen) and the p24 antigen assay for HIV-1, have been able to reduce the diagnostic window period. Methods Currently, blood donor screening for HBsAg, anti-HBc, anti-HCV, anti-HIV-1/2 and syphilis antibodies is mandated by the German authority (the Paul Ehrlich Institute). All blood donations must also be screened for HCV and HIV-1 using MP-NAT. The German Red Cross Blood Donor Services Baden-Württemberg-Hessen has implemented additional blood donor screening for anti-CMV (for platelet products from repeat donors) and MP-NAT screening for HBV, HAV and Parvovirus B19 on a voluntary basis. Results The epidemiology data within the last 4 years have found that 98% and 89% of hepatitis B and hepatitis C positive donors belonged to the first time donor population. Between 1997 and 2005, the German Red Cross Blood Donor Services detected hepatitis B, hepatitis C and HIV-1 in 43, 23 and 7 donors, respectively, using NAT. About 50% (22 out of 43) of these HBV-infected donors were in the acute phase of infection, while 21 were in the occult phase. Improvements in NAT technology, especially the introduction of full-automated extraction robot systems (e.g. the Zelos × 100), have been able to reduce the diagnostic window period, especially for hepatitis B, by improving the 95% level of detection. Nevertheless, five cases of NAT failures in detecting HIV-1 have been reported to the Paul-Ehrlich-Institute within the last decade. In 2010 in particular, three cases occurred in Germany due to mutations in the primer and probe binding regions. These cases restarted a discussion about risk analysis for NAT screening tests and serology methods in general. Conclusion In Germany, blood donor screening is performed using parallel serological assays (antigen and antibody detection) and by MP-NAT for hepatitis B, hepatitis C and HIV-1. The risk of false-negative test results due to mutations in primer and probe binding regions is higher for NAT systems than for antibody/antigen detection tests. Therefore, the manufactures of the NAT systems are advised to improve their systems by utilising amplification in at least two conserved regions (dual- or triple-targeting). The diagnostic window period for new screening strategies (e.g., antigen screening for HCV)     

Nucleic Acid Technology (NAT) testing for blood screening: impact of individual donation and Mini Pool – NAT testing on analytical sensitivity,screening sensitivity and clinical sensitivity

Globally, in a number of countries the donated blood is screened for serology markers for the Human immunodeficiency diseases‐1/2, the Hepatitis C virus (HCV), and the Hepatitis B virus (HBV). Several medium human development index (HDI) countries with reasonably high percentage of transfusion transmitted infections (TTI) are evaluating nucleic acid technology (NAT) assays to detect these viruses. Serology assays are performed on individual samples, while NAT is performed on either the individual donation (ID) or on a wide array of minipool (MP)‐NAT testing formats. The limit of detection of ID‐NAT assays equals the analytical sensitivity and that of pool testing reflects the pool size dependent decreased sensitivity also known as screening sensitivity. To many end users, it is not obvious that pool testing will have less sensitivity. In this review, mathematically predicted pool size dependent increase in risk days which is applicable to assays of all technologies is substantiated with published experimental results with NAT standards, clinical NAT only detected yields and detection misses by MP‐NAT. In the second half, the blood banking system in India, the donor base, and the variables in serology testing are discussed to explain the wide range of reported NAT yields at 1/300 to 1/17 753. Currently, NAT is not mandated in India, and the cost–benefit value of NAT is being seriously debated. As <5 IU/ml of HIV‐1 and HBV have resulted in TTIs, and nearly 83% of the HBV NAT yields in India have <20 IU/ml viral load, the most sensitive assay in the most sensitive format needs to be practiced to ensure maximum blood safety.     

Epidemiology of hepatitis B and C in Croatia

The incidence of HBV and HCV infection is hard to determine because of the high number of asymptomatic infections. According to data of the Croatian Institute of Public Health, there are 200 newly infected persons with hepatitis B and approximately the same number of newly identified HBsAg carriers occur each year. Accordingly, Croatia is among the countries with less than 2% of HBsAg carriers in the general population. In these circumstances, HBV infection is most often spread among adolescents and younger adults. The route of transmission is most often sexual (semen) or through the skin in high-risk groups. An increased risk of infection is found in newborns of HBsAg positive mothers, i.v. addicts, promiscuous individuals, male homosexuals, person in close contact with acutely ill or chronic HBsAg carriers, persons that come in contact with blood and other potentially contaminated body fluids, dialysis patients, patients with multiple blood transfusions, patients with transplanted organ or tissue, patients treated for hematologic malignancies and hemophilia, and persons who undergo acupuncture, tattooing or piercing, or travel to areas with a high prevalence of HBV infection. The estimated prevalence of HCV infection marker (anti-HCV) in the Croatian general population is more than 1% and the number of yearly infected with hepatitis C reported to the Croatian Institute of Public Health is around 200 cases. The highest incidence is found in the 20-40 age groups at a high risk of infection by the use of drug injection. At risk are persons who received transfusion of blood or blood products prior to the availability of blood screening of voluntary blood donors.     

Use of seroconversion panels to estimate delay in detection of anti-human immunodeficiency virus antibodies by enzyme-linked immunosorbent assay of pooled compared to singleton serum samples

The transfusion of unsafe blood worldwide accounts for 5 to 15% of new human immunodeficiency virus (HIV) infections, most of which occur in sub-Saharan Africa. While developed countries now apply PCR testing of pooled samples, some developing countries still do not have universal screening policies. More efficient low-cost procedures for the screening of pooled samples have the potential to encourage mass screening efforts in resource-poor settings. The aim of this study was to estimate the delay in the detection of HIV antibodies in pooled serum samples compared to that in singleton serum samples by enzyme-linked immunosorbent assay (ELISA) and to evaluate the risk of transfusion-transmitted HIV infection during the window period. Serial blood samples obtained from five HIV seroconversion panels were mixed with HIV-seronegative blood samples to create pools of 6, 12, 16, 24, 32, and 48 samples. The delay in detection of the first anti-HIV antibody-positive sample in tests with pooled samples was calculated for each pool size and compared to that obtained by testing of singleton samples and statistically evaluated by a robust log-linear regression analysis. The risk of a false-negative (FN) result caused by dilution was estimated by use of the incidence risk/window period model. The additional risk of transmission related to ELISA screening of pooled samples for HIV did not exceed 9% of the current risk of an FN result (estimated to be 1/1,067,000). The countries with virus prevalence rates in donors of less than 15% are expected to save up to 30% in the number of tests. ELISA screening of pooled samples could be considered in settings where the testing of blood supplies for HIV is not routinely done.