Transfusion Medicine
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REVIEW ARTICLE
Transfusion transmitted infections in thalassaemics: need for reappraisal of blood screening strategy in India V. Shyamala Research Diagnostics, Inc., Bengaluru, India Received 19 November 2013; accepted for publication 3 February 2014
SUMMARY The aim of the study was to assess the blood safety in India through prevalence in thalassaemic population. Safety of the blood supply is a subject of great concern for all recipients. This review attempts to assess the relevance and format of tests for viruses in the context of transfusion transmitted infection (TTI) prevalence in India. Serological marker testing for human immunodeficiency virus-1/2 (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is mandatory in India. Numerous TTI incidents in the repeat recipients supported by results from nucleic acid technology (NAT) testing indicate the deficiencies in blood safety. The β-thalassaemic population (3–17%) in India has been used to reflect on blood safety. The prevalence of HIV-1/2, HCV and HBV in the Indian donor population, the limitations in accessing safe donors, quality of serological tests and the impact on repeat recipients is evaluated. The reports point to prevalence of ∼2% of viral diseases in the blood donor population, and the insufficiency of serology testing resulting in up to 45% TTIs in thalassaemics. The revelation by individual donation (ID) NAT testing, of 1 per 310 units being serology negative-NAT reactive is alarming. Extrapolating the serology negative NAT reactive yields, for an annual blood supply of 7·9 million units, 23 700 units or nearly 100 000 blood components are likely to be infectious. Though the cost for ID-NAT testing is considered unaffordable for a medium development country such as India, the enormity of TTIs will place an unmanageable cost burden on the society. Key words: nucleic acid technology testing, occult infections, repeat recipients, serology markers, ß-thalassaemics, transfusion transmitted viral infections in India, window period.
BACKGROUND Blood safety is determined by detection and removal of infectious units from the blood supply. Globally the most practiced method
Correspondence: Venkatakrishna Shyamala, PhD, Research Diagnostics, Inc., 46/1, Palace Loop Road, Vasanth Nagar, Bangalore 560052, India. Tel.: +91 8971957545; fax: +91 8971957545; e-mail: [email protected]
© 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
of screening donated blood for human immunodeficiency virus (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is the detection of serology markers. Typically these markers are antibodies to HIV-1/2 or HCV, which reflect the immune response of the human body to infection, and the viral protein hepatitis B surface antigen (HBsAg) for HBV. In the course of infection the presence of viral proteins such as HBsAg, p24 for HIV-1, core protein for HCV are earlier events, and the presence of antibodies a much later event (Kleinman et al., 2009). Though there are combinational assays which integrate antibody detection with viral protein markers these tests are expensive and therefore less commonly used. Window period (WP) in the context of donor screening refers to the interval between the time the virus is present in blood and the time of a given marker detection, be it antibody, antigen or nucleic acid. The greatest threat to the safety of the blood supply using serology markers for screening is of seronegative donors during the infectious WP and, for HBV also the occult period. The safety of blood supply following any screening protocol can be evaluated by lookback and traceback analysis (Allain et al., 2013). The lookback analysis is triggered by a donor turning screening assay positive in the index sample and examining the recipients of previous donations from this donor. Such donors may present new or incident infections, indicating the change in prevalence of infection over time. Incidence estimates are calculated by testing a cohort of individuals at two different time periods for new infections. This strategy is difficult to implement due to the need to locate individuals for follow-up testing. Globally in countries with repeat donor base nearly all of the transfusion transmitted infections (TTIs) are realised through lookback analysis (Kleinman et al., 2009; Shyamala, 2014). However, in countries such as India, where donor registry is not maintained, and repeat donor base is negligible, lookback analysis is not possible. The traceback analysis is triggered by a recipient turning TTI positive, requiring traceback of the donor. In countries which have a donor registry, and sample archival system, recipient initiated donor traceback is possible. However, to date only a handful of infections are realised through traceback path (Shyamala, 2014). In India, as sample archival system is non-existent, pre- and post-transfusion monitoring of the recipient is mired in legal issues, and long term follow-up needed for hepatitis TTIs does not exist, traceback analysis is impossible. First published online 7 March 2014 doi: 10.1111/tme.12110
80 V. Shyamala A direct estimation of risks associated with a given blood screening protocol is to study the rate of infection prospectively in transfusion recipients. However, considering the low prevalence of TTI markers, and for individuals receiving occasional transfusion, data from an enormously large number of recipients has to be gathered, and this is impractical. The alternative is to analyse the prevalence and incidence of TTIs in the group of repeat recipients, as through multiple transfusions they serve as a microcosm reflecting blood safety status. Indeed, such an analysis of infections in recipients can be a better indicator of TTIs, as the prevalence data based on a multitude of screening assays have variable sensitivity and specificity. However, the prevalence in repeat recipients is also fraught with a different set of variables such as the recipient’s immunological ability to clear the disease, and the screening assay artifacts of the recipient sample. One such group of repeat recipients is the renal and haemodialysis group of patients, but because of various transfusion unrelated factors such as familial and other exposures, inability to mount robust immune response, the contribution of dialysis circuits, and advanced age, the association of infection exclusively to transfusion is difficult. A second group of repeat recipients are the haemophiliacs. However, they and only red cell fraction of blood, and only require recombinant factors and/or fractionated plasma factors, which in addition to the mandatory screening tests are also treated by viral reduction protocols. Hence haemophiliac repeat recipients might under-represent the viral infectivity of blood and blood products. A third group who can serve as a model to understand the status of blood safety is of thalassaemics who are provided packed red blood cells (PRBCs) or whole blood. The exclusive correlation of TTIs with transfusion in thalassaemics is possible, as they require transfusion from their first year of birth, and also because of their young age other means of acquiring infections can be excluded. The emphasis of this review is on the relevance of thalassaemics in India as the sentinel population to learn of blood safety, and the limitations, such as donor base, and screening assays in estimating the prevalence. The review focuses on recent publications such that the comparative screening tests are similar and relevant to current situation. The aim here is not to elaborate on the ramifications of TTIs due to compromised health of the thalassaemics.
THALASSAEMIA GENETICS, SYMPTOMS AND PREVALENCE Thalassaemia is a group of haemoglobin (Hb) disorders resulting in chronic haemolytic anaemia, and is of the following types: major and intermedia, sickle cell and HbE (Weatherall, 2010). The name of thalassaemia goes by the type that is underproduced. β-Thalassaemia indicates decreased synthesis of β chain, and α thalassaemia of decreased α chain. Unlike sickle cell Hb (HbS) where a single mutation is the cause, any mutation in the gene resulting in a defective protein can result in thalassaemia. With over 242 known mutations Transfusion Medicine, 2014, 24, 79–88
the occurrence of thalassaemia at 1·5% of global population (80–90 million people) is very high (Sinha et al., 2011). Worldwide α thalassaemia is the single most common gene disorder. β-Thalassaemia is most common in Southeast Asia, and the Mediterranean, with an estimated 63 000 children born globally each year. α-Thalassaemia occurs when one or more of the four genes, two on each of chromosome 16 are affected, and β-thalassaemia if one or both genes one on each of chromosome 11 are affected. For thalassaemias, depending on the defect the symptoms range from silent carriers requiring no transfusion to chronic transfusions (Weatherall, 2010). With repeat transfusion as the criterion to determine TTIs, the model of β thalassaemia major has been researched extensively to understand the degree of safety offered by the mandated screening technologies. The thalInd database is a repository of India thalassaemic information (Sinha et al., 2011). In India 64 β-globin mutations have been reported with IVS1-5C being the most common followed by the 619 bp deletion (Panigrahi & Marwaha, 2007; Verma et al., 2011). The general incidence of thalassaemia trait in India varies between 3 and 17% (Shah et al., 2010). With 100 000 β-thalassaemia patients, and 9000 thalassaemic annual births it is estimated that in India about 2 million units of PRBC are required for thalalssemic needs (Marwaha, 2010). It has been suggested that in the near future β-thalassaemia and related disorders are likely to emerge as the category of genetic disease that will have the most widespread impact on public health and health resources in India (Weatherall, 2010)
TTI COMPLICATIONS IN THE TREATMENT OF THALASSAEMICS The International Classification of Diseases (ICD) is the World Health Organization’s (WHO’s) ‘standard diagnostic tool for epidemiology, health management and clinical purposes, and used to predict mortality and morbidity statistics’. The ICD reports indicate that globally blood-borne viral infections mainly hepatitis C and B are the second most common cause of death after heart failure in the general population. Worldwide thalassaemia patients exhibit 0·3–5·7% HBsAg and 4·4–85·4% HCV positivity. As very good solutions are currently available for iron chelation, the disease management has shifted to address hepatocellular carcinoma (HCC) through TTIs (Di Marco et al., 2010). Typically 15% of acute HCV infections found in first time donors, and 54% in repeat donors have been shown to resolve spontaneously. Of the remaining, 85% of the serology negative donors will remain a source of occult infection. Generally 54–86% of acute HCV cases progress to chronic hepatitis, 25% of chronic hepatitis cases progress to cirrhosis, and 1–4% lead to HCC (Mukhopadhya, 2008). HBV has a unique disease pattern, in that the chronic carriers remain infectious throughout their life, albeit intermittently (Fig. 1; Vermeulen et al., 2012). In thalassaemics, as iron overload affects heart and liver, HCV and HBV infections add another layer of complexity to liver functionality. Thalassaemia major or intermedia subjects have been reported to exhibit HCC at a young age of 45 years © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
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compared with 66 years in the control group (Di Marco et al., 2010). The rate of progression of HIV-1 disease is also known to be faster in iron overload conditions. The early acquisition of infection and rapid disease progress is also ascribed to compromised immune status in thalassaemics. Although the above details are specific for thalassaemics, endemic proportion of diabetes (5% prevalence in the 20–79 age group) and heart problems (12% national prevalence) indicates the less than optimal health status of the Indian population.
initial serology reactive units. Several blood banks use rapid tests as supplemental to screening tests, which a decade ago had similar sensitivity as the screening ELISA test. However, over the years much improvement has been made in advancing the sensitivity and specificity of the screening ELISA/CIA tests and consequently rapid tests have become comparatively less sensitive (Laperche, 2013). The use of less sensitive rapid tests as supplemental test has caused much confusion regarding the accuracy of reactive screening results.
EFFORTS TO LIMIT TTI THROUGH BLOOD SCREENING IN INDIA
HBV prevalence in blood donors and contributing factors
Through the establishment in 1987 of National AIDS Control Organization (NACO), the Drug Controller General, India (DCGI) is vested with the responsibility to approve licensing of blood and blood products in 1992, and in 2002 by formulating a National Blood Policy, efforts have been made to make the blood supply safer in India (Regulatory requirements of blood and /or its components including blood products, http://cdsco.nic.in/html/guideline.htm; NACO, 2007). Testing of donated blood for anti-HIV was mandated in India in 1989, for HBsAg in 1992 and for anti-HCV in 2001 (NACO, 2007). However, as detailed in this article effective surveillance system which is required for realistic predictions of prevalence for effective public health policy for measures to curtail increase in infection is still lacking. Of the about 2700 blood banks nation-wide, nearly 40% are in the government category, 35% in private and 25% in private charitable categories (Choudhury, 2011). Depending on the category of blood bank there is a wide range in the quality of serology assays. The private hospitals and private charitable hospitals choose either third or fourth generation enzyme-linked immunosorbent assays (ELISAs) or chemiluminescent immunoassays (CIAs) that meet their internal quality and affordability criterion. A majority of government hospitals are provided third generation ELISA serology assay reagents free of cost, but of variable quality. A number of blood banks that are resource and facilities constrained, and also have very few donations are permitted to use rapid tests to screen (NACO, 2012). Using reagents of diverse quality and heterogeneous sample groups, in India the prevalence in general population is reported to be 0·36% for HIV-1/2, 0·09–7·89% for HCV, 2·5–4% for HBV (NACO, 2008–2009; Mukhopadhya, 2008; Abraham, 2012). Amongst the blood donor population including both volunteer and family/replacement donors the HIV-1/2 prevalence is 0·17–0·3% (Malhotra et al., 2013; NACO, 2008–2009), 0·4% HCV and 1·2% HBV (NACO, 2008–2009; Abraham, 2012). Subject to regional life-style habits, religious beliefs and nonrecommended practices a wide range in prevalence numbers have been recorded. In practice not all blood banks test the initial reactives in two replicates with the same reagent (which in itself does not control for assay specificity), and even fewer labs test repeat reactives with a confirmatory supplemental second assay. In India the majority of blood banks discard the © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
On the basis of HBsAg prevalence, India is considered an intermediate HBV endemic country. At a prevalence of up to 4% in the general population, and a disease burden of >50 million, India has the second largest pool of HBV carriers in the world (Abraham, 2012). Additionally 80% of HCC in India is virus associated, with 36–43% ascribed to HBV and 30% to HCV (Abraham, 2012). In India blood screening for HBsAg as the only marker for HBV was implemented in 1992. HBsAg is a direct measure of the viral protein marker, and depending on the reagent this can be detected as early as 38 days in plasma (Kleinman et al., 2009; Fig. 1). Through the presence of a 1000- to 10 000-fold excess of non-DNA containing sub viral particles, there is a relatively high concentration of HBsAg, resulting in early detection (Gerlich et al., 2007). HBsAg being a viral protein is a transient marker lasting only 63 days of infection in acute infections, which is a drawback (Vermeulen et al., 2012). Despite HBsAg being a viral protein and the anticipated high specificity, using the most advanced reagents 56% of repeat reactives could not be confirmed by a second neutralisation reagent assay (Stramer et al., 2013b). The use of less sensitive HBsAg assays and HBsAg mutations that are not detected by the antibodies allow the infection to proceed to occult carrier stage (Stramer et al., 2011). Occult B infection (OBI) is characterised by the absence of HBsAg, presence of anti-HBc with or without the presence of anti-HBs, and intermittently detected extremely low HBV viral load (VL), and VL as low as 5 IU mL−1 DNA has been shown to cause TTIs (Allain et al., 2013). Many of the thalassaemia prevalent countries also have high HBV occult carrier population, and in India it is estimated at 19% (Abraham, 2012). In studies from Northern India up to 10% of the blood donors were anti-HBc positive with 0·15–1·5% of these being DNA positive and hence potentially infectious (Makroo et al., 2012; Sen & Arora, 2012). In a study from Southeastern India 30% of the HBsAg negative donors were anti-HBc positive, and of them 30% were HBV DNA positive (Panigrahi et al., 2010). In a 2013 study from the United States anti-HBc positive donors (29 909) were 10 times more prevalent than HBsAg positives (2703), and 2·5% (723) of OBI repeat reactives were HBV DNA positive (Stramer et al., 2013b). Anti-HBc screening is mandatory in the United States and reactive units are excluded. In India through the use of HBsAg non-reactive OBI units, the transfusion risk of infectious HBV units remains high. While the screening for anti-HBc would identify nearly all chronic carriers, Transfusion Medicine, 2014, 24, 79–88
82 V. Shyamala
Fig. 1. Serum markers and disease status of HBV infection. Profile of markers in acute resolving hepatitis B infection (top); chronic hepatitis B infection (Bottom). (Courtesy, Prof. J. P. Allain, University of Cambridge, UK.)
in India it also would result in high discard rate and donor deferral. In a system that is already burdened with insufficient blood supply the discard of non-infectious OBI units would be difficult to accommodate. The prevalent HBV genotypes in India are predominantly D, followed by A, C and additional recombinant forms in the Eastern region (Ghosh et al., 2013). A few of the HBV genotypes such as genotypes D and A2 do not reach high VLs, remaining below detection limit, and result in increased possibilities of infectious unit transfusions (Allain et al., 2013). The information regarding genotypes could also be skewed, as low VL samples are a challenge to amplify for genotype analysis. In India donors with HBsAg reactivity are referred to the gastroenterology departments and associates in hospital-associated blood banks and stand-alone blood banks, respectively.
HCV prevalence in blood donors and contributing factors The typical blood screening assay for HCV is for detecting antiHCV antibodies, and was mandated in India in 2001. Antibodies remain in circulation for a very long time lasting years, even after viral remission, hence antibody reactivity results suggest either a current HCV infection, a past infection or false positivity. As the HCV antibodies can be detected only after 65–70 days of WP, HCV has the longest period of non-detection of infectious units (Kleinman et al., 2009). The delayed immune response means that the viral propagation proceeds unchecked and remains high during this interval. The third generation anti-HCV ELISA and CIA assays are known to have high false positivity. In a study from the United States only 21–34% of repeat reactives could be confirmed by a second reagent Western blot (WB) recombinant immunoblot assay (RIBA) (Stramer et al., 2013a). Amongst the Indian blood donors the initial reactivity as detailed below was Transfusion Medicine, 2014, 24, 79–88
reported to be between 0·2 and 0·77%. Of the 0·2% (140) initial reactives, 63% (87) were non-reactive by a second ELISA reagent, and all of these were negative by confirmatory RIBA and for RNA (Tulsiani et al., 2012). In another study the initial reactive rate was 0·77%, and only 70% tested positive by a second assay and 21% positive by confirmatory immunoblot assay (Raghuraman et al., 2003b). Combination assays of HCV antibody and core protein viral antigen which can reduce the WP by 21 days are not widely used because of cost (Laperche et al., 2005). The availability of the confirmatory RIBA WB has been inconsistent and currently discontinued (Stramer et al., 2013a). In summary, >30% of the initial reactive samples are not confirmed with a second reagent, and of the remaining positives 50 transfusions resulted in 57% HCV prevalence. Age dependent breakdown of the prevalence was not provided to determine pre- and post-anti-HCV screening. From Western India where up to 7·48% thalassaemia is observed, using supplemental confirmatory assay 25% HCV prevalence has been reported (Jain et al., 2012). In a 3 year (1993–1995) long-term study by Choudhury et al. (1998) anti-HCV prevalence of 23, 30·7 and 35·9%, was observed for each consecutive year. In contrast, a prevalence of 2% was reported in another study involving 200 thalassaemics, again with no increase in TTIs with increased transfusions (Vidja et al., 2011). In order to understand if the high HCV prevalence of up to 57% in thalassaemics was unique to India or if it was a phenomenon also applicable to other thalassaemia endemic countries, publications from a few other countries were reviewed. The information in Table 1 points to the high prevalence of HCV in thalassaemics in all countries. In all of the above studies a breakdown of pre- and postserology screening results has not been provided. Because of the unique profile of HCV with very long WP for antibody development it is critical to analyse the data for pre- and postserology screening periods to gauge the impact of serology screening. In the only study providing a breakdown, Shah et al. (2010) indicate that 15% (six) of the children born after serology screening initiation were HCV positive. Overall 45% HCV prevalence was observed in this study, with double infections of HCV–HIV; and one triple infection for HBV also. Although Transfusion Medicine, 2014, 24, 79–88
they did not perform a comparative donor testing, the high prevalence in thalassaemics attests to infectivity of serology negative units. Despite the wide variance the high prevalence of HCV has led to a consensus call in all the publications for improved pretransfusion screening, including the suggestion to adopt nucleic acid technology (NAT) testing.
HIV positivity in thalassaemics in India Implemented in India in 1989, at 0·3% prevalence in blood donors HIV-1/2 has the lowest prevalence of the three viruses. In comparison a 2% HIV prevalence in thalassaemics of ages 8, 11 and 12 years was observed, suggesting TTI through serology qualified units (Shah et al., 2010). All three patients were coinfected with HCV, again pointing to transfusion as the cause. In a 200 patient one time study with a minimum transfusion of 10 units of blood, six (3%) HIV infections were observed in the 0–20 age group, again with all of them from postserology screening period (Vidja et al., 2011). Similarly in a 3-year annually monitored study of 39 patients of 3·5–9 years; one instance (2·5%) of HIV was observed (Choudhury et al., 1998). In a study with 126 patients of the age group 10 months to 22 years 4% HIV prevalence was observed. Although the study does not provide a breakdown of pre- and post-anti-HIV screening incidences, it is likely that a majority of the patients received serology negative units (Mathur et al., 2008). In another study with 96 patients for 2 years monitored at 6-month intervals a 1% prevalence of HIV was observed (Jain et al., 2012). Of the three viruses through early implementation it is possible to evaluate the impact of serology screening for HIV. The consensus of 1–4% prevalence in thalassaemics versus the observed 0·3% in donors points to the failure of serology assays to adequately interdict infectious HIV units. Through repeat transfusions, thalassaemic recipients indicate vulnerability to heterogeneous strains, resulting in recombinations and mutations of HIV virus.
NEED FOR NAT TESTING In spite of the mandated serology assays in India the reports for all three viral TTIs indicate the inadequacy of serology-only screening in ensuring blood safety. The abundant literature for post-serology infections for HIV and HBV and limited information for HCV definitely point to the necessity of rethinking about the sufficiency of mandated serology tests alone. Indeed a majority of the publications recommend the use of NAT testing. By targeting the very early event in viral propagation through the detection of viral genomes which are the source of infection, NAT tests provide the shortest WP. The most sensitive NAT tests amplify the nucleic acid several million folds to detect the presence of very low number of copies of viral NA. The current technologies are so fine-tuned for NA isolation, amplification and detection that the WP from the day of presence of virus in blood to the day of detection depends on the virus © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
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Table 1. Prevalence of anti-HCV serology marker in general population, blood donors and thalassaemics
Country
Serology testing since
% HCV prevalence General population
India
2001
1–1·9 (Sievert et al., 2011)
Iran Pakistan
1996 2003
Malaysia
1995
0·3–1 (Azarkeivan et al., 2012) 4·7 (Waheed et al., 2009; Sievert et al., 2011) 1·5–2·3 (Duraiswamy et al., 1993)
Thailand Egypt
1991 1999
2·8 (Sievert et al., 2011) 15 (Sievert et al., 2011)
Blood donors
Thalassaemics
0·4 (NACO, 2008–2009; Abraham, 2012) 0·12 (Azarkeivan et al., 2012) 0·5–8·9 (Waheed et al., 2009; Sievert et al., 2011) 0·4 (Haslina et al., 2012)
16–26 (Mukhopadhya, 2008; Abraham, 2012) 19 (Di Marco et al., 2010) 48·6 (Waheed et al., 2009; Di Marco et al., 2010) 13–22 (Lee et al., 2005; Di Marco et al., 2010) 21·2 (Di Marco et al., 2010) 24–76 (El-Faramawy et al., 2012; El-Shanshory et al., 2013)
0·31–3·54 (Sievert et al., 2011) 8–14·7 (Sievert et al., 2011; El-Shanshory et al., 2013)
doubling time. Assuming that infectivity first occurs with one viral copy in 20 mL plasma (mL of plasma per unit of PRBC), based on results from seroconversion panels infectious WP days have been calculated (Kleinman et al., 2009). When practiced in the highly sensitive individual donation (ID) NAT testing format, with an analytical sensitivity for HCV at eight copies per millilitre (1 IU = 2·5 copies), with a doubling time of 0·45 days, the infectious unit is detected within three WP days, reducing the infectious risk period by 58 days from third generation ELISA anti-HCV serology testing. To date transfusion of several million units of blood qualified by ID-NAT testing has not resulted in a single case of HCV transmission. For HBV through ID-NAT testing format, with an analytical sensitivity of 12 copies per millilitre (1 IU = 5·6 copies), with a 2·5-day doubling period of VL, the infectious unit is detected within 15 WP days, reducing the infectious risk period by 21 days over HBsAg serology testing. To date for several million units tested by ID-NAT there is only one instance of TTI caused by HBsAg negative WP unit with 1·5 copies per millilitre of HBV DNA (Vermeulen et al., 2012). Globally, because OBI serology marker screening is impractical, OBI transmissions can be interdicted only through sensitive NAT screening. ID-NAT testing for HIV-1, with an analytical sensitivity of 16 copies per millilitre (1 IU = 0·6 copies), with a doubling time of 0.85 days detects infectious unit within 6 WP days, reducing the infectious risk WP by 13 days over anti-HIV serology testing. To date the release of millions of ID-NAT tested units has resulted only in a single case of HIV transmission with 3–4·5 Cps mL−1 (Salles et al., 2013). The single case each of HBV and HIV, are reminders that while ID-NAT testing offers the maximum safety, it still does not provide risk-free blood. ID-NAT is the most sensitive format because each sample is tested individually, and directly reflects the analytical sensitivity of the assay. A second method of practice is the pooled testing which introduces an element of dilution, with decrease in analytical sensitivity proportional to the pool size. This is also referred to as screening sensitivity, and also results in decreased clinical sensitivity. In the past a number of countries practiced pooled testing format for a variety of reasons including: (i) very limited option of automation for high through-put required © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
for individual unit testing, (ii) high cost for each result, (iii) challenges in implementing complex technology, (iv) concerns of amplicon contamination through NA amplification of a large number of samples, (v) insufficient proof of the benefit of IDNAT, (vi) low prevalence in advanced countries coupled with (vii) superior quality and additional serology assays resulting in insignificant cost-benefit. Currently, these factors have been addressed through the possibility of high-throughput screening and automation, resulting in ease of implementation, and closed systems to control contamination. Most importantly, experience with NAT testing in the past 15 years has led to important revelations about the need for ID-NAT in moderate prevalence countries. Several retrospective studies have demonstrated the correlation of ID-NAT with detection of viruses in the previously missed pooled units, and the estimates of TTIs that could have been interdicted by ID-NAT (Shyamala, 2014). All these factors have led a large number of countries to adopt ID-NAT testing. Additionally, HBV DNA screening as an indicator of infectivity is more effective in countries with high to medium endemicity and where anti-HBc is not mandated. Currently in India 7% of the 7·9 million units of annually donated blood is screened by ID-NAT testing. The results from a multicenter study indicated serology negative NAT reactive yields to be in the range of 1 per 1333 (nine yields for 12 000 units) with six of nine yields being HBV (Makroo et al., 2008). Being a multicenter study the serology tests used to define NAT yields were a mix of third and fourth generation assays from a variety of vendors. In another study with 73 898 tests with advanced serology tests, a NAT yield of 1 per 610 donations was observed; with 60% being HBV, 30% being HCV, 0.8% being HIV-1, and 8% being double infection HBV and HCV (Agarwal et al., 2013). Using this same donor base and an improved HBV detecting NAT assay HBV NAT yield of 1 per 301 was reported (Chatterjee, 2012). The improved HBV reagent detected higher percentage of OBI cases, with >80% having very low VL of 62% being HBV yields. Nearly 70% of the HBV NAT yields are OBI cases, which being HBsAg negative are currently being transfused as ‘safe blood’. While, whole blood, particularly fresh whole blood (stored for less than a week) remains the product of choice for emergency bleedings, the primary transfusion need of thalassaemics is for PRBCs. However, in India only 30% of the blood banks have component preparation facilities (Marwaha, 2010). As a result, at many facilities, thalassaemics and general recipients irrespective of their specific need for PRBC or platelets are provided with whole blood. While there are several immunohematology reasons to discontinue this practice, there are also compelling TTI reasons to discontinue this practice. Nearly all of the HIV1, HCV and HBV VL in the blood is present in the plasma fraction. It is estimated that one unit of PRBC has 20–30 mL of plasma, and one unit of platelets has 50 mL of plasma versus a unit of whole blood which contains 180–220 mL plasma. Hence the whole blood transfusion results in six to seven fold excess over PRBC. The total VL transfused has been shown to be directly correlated to cases of TTI (Kleinman et al., 2009; Allain et al., 2013). There are numerous reports of TTIs with plasma transfusion, while PRBC and platelets from the same unit have not resulted in TTI. Hence in all situations TTIs can be minimised through the required use of components.
CONCLUSION The true value of diagnostics depends on assays that are early predictors of infection offering maximum blood safety. However, through breach in ‘safe blood transfusion’ thalassaemics are confronted by new and more severe health challenges of TTIs contributing to increased mortality, and this can be extrapolated to general recipients as well. It can be undoubtedly stated that through the current serology screening practices, TTIs for HIV-1, HCV and HBV are proceeding unchecked. Despite official positions, the approach of exclusive volunteer non-remunerated safe donors and vigilant donor deferral is not an immediately implementable way to curtail this
REFERENCES Abraham, P. (2012) Viral hepatitis in India. Clinical Laboratory Medicine, 32, 159–174. Agarwal, N., Chatterjee, K., Coshic, P. & Borgohain, M. (2013) Nucleic acid testing for blood banks: an experience from a tertiary care centre in New Delhi, India. Transfusion and Apheresis Science, 49, 482–484. Allain, J.P., Mihaljevic, I., Gonzalez-Fraile, M.I. et al. (2013) Infectivity of blood
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increase. From a blood supply point of view, family donors are a motivated population that significantly contributes to the Indian blood supply and cannot be deferred without due consideration of the evidence. Transforming the donor base to voluntary repeat donors is a sociological change which will take a long time to accomplish. For the immediate, implementable testing practice for maximum safety is the solution. Although the argument of unaffordability of ID-NAT testing has been the premise for pooled testing, the residual risk of the resulting TTIs places a serious burden on the health care, finances, society and human life. True health care costs applicable to Indian scenario have not been available. The cost of advanced stage HIV-1 treatment in Thailand has been projected at USD $1158 (van Hulst et al., 2009). As emphasized in the review by Shyamala (2014), in India the cost is substantially borne by the government and this expense could be decreased by improved blood screening to prevent potential TTIs. The situation for hepatitis is even grimmer, as the clinical symptoms of the infection take a long time to manifest and the magnitude of the disease burden will not be apparent for years to come. Additionally, unlike for HIV, there is no national policy for patient treatment for hepatitis. The economic burden of HBV/HCV infection is substantial even in the early stages of infection, and escalates steeply with advanced stages of infection. The annual cost of weekly injectable pegylated interferon therapy available for hepatitis is billed at INR 884 000 (USD $15 000). The treatment costs for HCC and decompensated cirrhosis in Thailand are estimated at USD $6760 and USD $13 973, respectively; these costs are the highest in the health care management (van Hulst et al., 2009). These observations are a wake-up call for providing additional layer of security to national blood supply through NAT testing.
ACKNOWLEDGMENT The concept, literature review and writing was solely by the author. The author likes to thank Mr. Sangeet Kini for providing valuable background information.
CONFLICT OF INTEREST V. S. is a part-time consultant for Novartis Diagnostics.
products from donors with occult hepatitis B virus infection. Transfusion, 53, 1405–1415. Azarkeivan, A., Toosi, M.N., Maghsudlu, M., Kaiabad, S.A., Hajibeigi, B. & Hadizadeh, M. (2012) The incidence of hepatitis C in patients with thalassemia after screening in blood transfusion centers: a fourteen-year study. Transfusion, 52, 1814–1818. Center for Disease Control and Prevention (CDC) (2013) Detection of acute HIV
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© 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
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Jaiswal, S.P.B., Chitnis, D.S., Jain, A.K., Inamdar, S., Porwal, A. & Jain, S.C. (2001) Prevalence of hepatitis viruses among multi-transfused homogenous thalassemia patients. Hepatology Research, 19, 247–253. Kaur, R., Basu, S., Kaur, G. & Singh, D. (2011) Fourth generation enzyme immunoassays for screening of HIV in blood donors: need of the hour. Indian Journal of Pathology and Microbiology, 54, 433–434. Khakhkhar, V. & Joshi, P.J. (2006) HBsAg seropositivity among multi-transfused thalassemic children. Indian Journal of Pathology and Microbiology, 49, 516–518. Kleinman, S.H., Lelie, N. & Busch, M.P. (2009) Infectivity of human immunodeficiency virus-1, hepatitis C virus and hepatitis B virus and risk of transmission by transfusion. Transfusion, 49, 2454–2489. Kumar, R. & Kaur, A. (2013) Importance of blood screening by nucleic acid testing with a repeat testing algorithm in hepatitis C and B virus high-prevalence region. Vox Sanguinis, 105 (Suppl. 2), 86. Laperche, S. & on behalf of the Francophone African group for Research in Blood transfusion (2013) Multinational assessment of blood-borne virus testing and transfusion safety on the African continent. Transfusion, 53, 816–826. Laperche, S., Le Marrec, N., Girault, A. et al. (2005) Simultaneous detection of Hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection. Journal of Clinical Microbiology, 43, 3877–3883. Lee, W.S., Teh, C.M. & Chan, L.L. (2005) Risks of seroconversion of hepatitis B, hepatitis C and human immunodeficiency viruses in children with multitransfused thalassemia major. Journal of Paediatrics Child Health, 41, 265–268. Makroo, R.N., Choudhury, N., Jagannathan, L. et al. (2008) Multicenter evaluation of individual donor nucleic acid testing [NAT] for simultaneous detection of human immunodeficiency virus-1 and hepatitis B & C viruses in India blood donors. Indian Journal of Medical Research, 127, 140–147. Makroo, R.N., Chowdhry, M., Bhatia, A., Arora, B. & Rosamma, N.L. (2011) Prevalence of HIV among blood donors in a tertiary care centre of North India. Indian Journal of Medical Research, 134, 950–953. Makroo, R.N., Chowdhry, M., Bhatia, A., Arora, B. & Rosamma, N.L. (2012) Hepatitis B core antibody testing in Indian blood donors: a double-edged sword!. Asian Journal of Transfusion Science, 6, 10–13.
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Malhotra, S., Marwaha, N. & Saluja, K. (2013) Seroprevalence of human immunodeficiency virus in North Indian blood donors using third and fourth generation enzyme linked immunosorbent assay. Asian Journal of Transfusion Science, 7, 125–129. Marwaha, N. (2010) Whole blood and component use in resource poor settings. Biologicals, 38, 68–71. Mathur, M., Wanjari, K. & Turbadkar, D. (2008) Surveillance of HIV, hepatitis C and hepatitis B in multitransfused thalassemics. Indian Journal of Medical Microbiology, 26, 205–206. Mukhopadhya, A. (2008) Hepatitis C in India. Journal of Biosciences, 33, 465–473. National Aids Control Organisation Ministry of Health and family Welfare (2007) Standards for Blood banks and Blood Transfusion Services. Government of India, New Delhi. National Aids Control Organisaton, Department of AIDS Control, Ministry of Health & Family Welfare, Government of India. Computerised Management Information Systems (CMIS) bulletin (April 2008- March 2009) [WWW document]. URL http://aidsdatahub.org/en/indiareference-library/item/12112-annual-cmisbulletin-2008-09-national-aids-controlorganisation-india-2010 (Accessed 10/10/13) Panigrahi, I. & Marwaha, R.K. (2007) Mutational spectrum of thalssemias in India. Indian Journal of Human Genetics, 31, 36–37. Panigrahi, R., Biswas, A., Datta, S. et al. (2010) Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in Southeastern India: implications for transfusion. Virology Journal, 7, 204–210. Raghuraman, S., Shaji, R.V., Sridharan, G., Radhakrishnan, S., Chandy, G., Ramakrishna, B.S. & Abraham, P. (2003a) Distribution of the different genotypes of HCV among patients attending a tertiary care hospital in South India. Journal of Clinical Virology, 26, 61–69. Raghuraman, S., Subramanian, T., Daniel, D., Sridharan, G. & Abraham, P. (2003b) Occurrence of false positives during testing for antibodies to Hepatitis C virus among volunteer blood donors in India. Journal of Clinical Microbiology, 41, 1788–1790. Salles, N.A., Levi, J.E., Barreto, C.C., Sampaio, L.P., Romano, C.M., Sabino, E.C. & Transfusion Medicine, 2014, 24, 79–88
88 V. Shyamala Mendrone, A. Jr. (2013) Human immunodeficiency virus transfusion transmission despite nucleic acid testing. Transfusion, 53, 2593–2595. Sen, A. & Arora, S. (2012) Impact of individual donor nucleic-acid amplification testing of blood donors in interdicting transmission of transfusion transmitted HIV, hepatitis B and hepatitis C in India. Vox Sanguinis, 103 (Suppl. 1), 148–149. Seth, P. (2010) Evolution of HIV-1 in India. Indian Journal of Virology, 21, 3–7. Shah, N., Mishra, A., Chauhan, D., Vora, C. & Shah, N.R. (2010) Study on effectiveness of transfusion program in thalassemia major patients receiving multiple blood transfusions at a transfusion center in western India. Asian Journal of Transfusion Science, 4, 94–98. Shrivastava, P. (2013) ID-NAT testing and the benefit of user algorithm in a standalone Indian blood bank. Vox Sanguinis, 105 (Suppl. 2), 83. Shyamala, V. (2014) Factors in enhancing blood safety by nucleic acid technology testing for human immunodeficiency virus, hepatitis C virus and hepatitis B virus. Asian Journal of Transfusion Science, 8, 13–18. Sievert, W., Altraif, I., Razavi, H.A. et al. (2011) A systematic review of hepatitis C epidemiology in Asia, Australia and Egypt. Liver International, 32, 61–80. Singh, H., Pradhan, M., Singh, R.L., Phadke, S., Naik, S.R., Aggarwal, R. & Naik, S. (2003) High frequency of hepatitis B virus infection in patients with β-thalassemia receiving multiple transfusions. Vox Sanguinis, 84, 292–299.
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REVIEW ARTICLE
Transfusion transmitted infections in thalassaemics: need for reappraisal of blood screening strategy in India V. Shyamala Research Diagnostics, Inc., Bengaluru, India Received 19 November 2013; accepted for publication 3 February 2014
SUMMARY The aim of the study was to assess the blood safety in India through prevalence in thalassaemic population. Safety of the blood supply is a subject of great concern for all recipients. This review attempts to assess the relevance and format of tests for viruses in the context of transfusion transmitted infection (TTI) prevalence in India. Serological marker testing for human immunodeficiency virus-1/2 (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is mandatory in India. Numerous TTI incidents in the repeat recipients supported by results from nucleic acid technology (NAT) testing indicate the deficiencies in blood safety. The β-thalassaemic population (3–17%) in India has been used to reflect on blood safety. The prevalence of HIV-1/2, HCV and HBV in the Indian donor population, the limitations in accessing safe donors, quality of serological tests and the impact on repeat recipients is evaluated. The reports point to prevalence of ∼2% of viral diseases in the blood donor population, and the insufficiency of serology testing resulting in up to 45% TTIs in thalassaemics. The revelation by individual donation (ID) NAT testing, of 1 per 310 units being serology negative-NAT reactive is alarming. Extrapolating the serology negative NAT reactive yields, for an annual blood supply of 7·9 million units, 23 700 units or nearly 100 000 blood components are likely to be infectious. Though the cost for ID-NAT testing is considered unaffordable for a medium development country such as India, the enormity of TTIs will place an unmanageable cost burden on the society. Key words: nucleic acid technology testing, occult infections, repeat recipients, serology markers, ß-thalassaemics, transfusion transmitted viral infections in India, window period.
BACKGROUND Blood safety is determined by detection and removal of infectious units from the blood supply. Globally the most practiced method
Correspondence: Venkatakrishna Shyamala, PhD, Research Diagnostics, Inc., 46/1, Palace Loop Road, Vasanth Nagar, Bangalore 560052, India. Tel.: +91 8971957545; fax: +91 8971957545; e-mail: [email protected]
© 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
of screening donated blood for human immunodeficiency virus (HIV-1/2), hepatitis C virus (HCV) and hepatitis B virus (HBV) is the detection of serology markers. Typically these markers are antibodies to HIV-1/2 or HCV, which reflect the immune response of the human body to infection, and the viral protein hepatitis B surface antigen (HBsAg) for HBV. In the course of infection the presence of viral proteins such as HBsAg, p24 for HIV-1, core protein for HCV are earlier events, and the presence of antibodies a much later event (Kleinman et al., 2009). Though there are combinational assays which integrate antibody detection with viral protein markers these tests are expensive and therefore less commonly used. Window period (WP) in the context of donor screening refers to the interval between the time the virus is present in blood and the time of a given marker detection, be it antibody, antigen or nucleic acid. The greatest threat to the safety of the blood supply using serology markers for screening is of seronegative donors during the infectious WP and, for HBV also the occult period. The safety of blood supply following any screening protocol can be evaluated by lookback and traceback analysis (Allain et al., 2013). The lookback analysis is triggered by a donor turning screening assay positive in the index sample and examining the recipients of previous donations from this donor. Such donors may present new or incident infections, indicating the change in prevalence of infection over time. Incidence estimates are calculated by testing a cohort of individuals at two different time periods for new infections. This strategy is difficult to implement due to the need to locate individuals for follow-up testing. Globally in countries with repeat donor base nearly all of the transfusion transmitted infections (TTIs) are realised through lookback analysis (Kleinman et al., 2009; Shyamala, 2014). However, in countries such as India, where donor registry is not maintained, and repeat donor base is negligible, lookback analysis is not possible. The traceback analysis is triggered by a recipient turning TTI positive, requiring traceback of the donor. In countries which have a donor registry, and sample archival system, recipient initiated donor traceback is possible. However, to date only a handful of infections are realised through traceback path (Shyamala, 2014). In India, as sample archival system is non-existent, pre- and post-transfusion monitoring of the recipient is mired in legal issues, and long term follow-up needed for hepatitis TTIs does not exist, traceback analysis is impossible. First published online 7 March 2014 doi: 10.1111/tme.12110
80 V. Shyamala A direct estimation of risks associated with a given blood screening protocol is to study the rate of infection prospectively in transfusion recipients. However, considering the low prevalence of TTI markers, and for individuals receiving occasional transfusion, data from an enormously large number of recipients has to be gathered, and this is impractical. The alternative is to analyse the prevalence and incidence of TTIs in the group of repeat recipients, as through multiple transfusions they serve as a microcosm reflecting blood safety status. Indeed, such an analysis of infections in recipients can be a better indicator of TTIs, as the prevalence data based on a multitude of screening assays have variable sensitivity and specificity. However, the prevalence in repeat recipients is also fraught with a different set of variables such as the recipient’s immunological ability to clear the disease, and the screening assay artifacts of the recipient sample. One such group of repeat recipients is the renal and haemodialysis group of patients, but because of various transfusion unrelated factors such as familial and other exposures, inability to mount robust immune response, the contribution of dialysis circuits, and advanced age, the association of infection exclusively to transfusion is difficult. A second group of repeat recipients are the haemophiliacs. However, they and only red cell fraction of blood, and only require recombinant factors and/or fractionated plasma factors, which in addition to the mandatory screening tests are also treated by viral reduction protocols. Hence haemophiliac repeat recipients might under-represent the viral infectivity of blood and blood products. A third group who can serve as a model to understand the status of blood safety is of thalassaemics who are provided packed red blood cells (PRBCs) or whole blood. The exclusive correlation of TTIs with transfusion in thalassaemics is possible, as they require transfusion from their first year of birth, and also because of their young age other means of acquiring infections can be excluded. The emphasis of this review is on the relevance of thalassaemics in India as the sentinel population to learn of blood safety, and the limitations, such as donor base, and screening assays in estimating the prevalence. The review focuses on recent publications such that the comparative screening tests are similar and relevant to current situation. The aim here is not to elaborate on the ramifications of TTIs due to compromised health of the thalassaemics.
THALASSAEMIA GENETICS, SYMPTOMS AND PREVALENCE Thalassaemia is a group of haemoglobin (Hb) disorders resulting in chronic haemolytic anaemia, and is of the following types: major and intermedia, sickle cell and HbE (Weatherall, 2010). The name of thalassaemia goes by the type that is underproduced. β-Thalassaemia indicates decreased synthesis of β chain, and α thalassaemia of decreased α chain. Unlike sickle cell Hb (HbS) where a single mutation is the cause, any mutation in the gene resulting in a defective protein can result in thalassaemia. With over 242 known mutations Transfusion Medicine, 2014, 24, 79–88
the occurrence of thalassaemia at 1·5% of global population (80–90 million people) is very high (Sinha et al., 2011). Worldwide α thalassaemia is the single most common gene disorder. β-Thalassaemia is most common in Southeast Asia, and the Mediterranean, with an estimated 63 000 children born globally each year. α-Thalassaemia occurs when one or more of the four genes, two on each of chromosome 16 are affected, and β-thalassaemia if one or both genes one on each of chromosome 11 are affected. For thalassaemias, depending on the defect the symptoms range from silent carriers requiring no transfusion to chronic transfusions (Weatherall, 2010). With repeat transfusion as the criterion to determine TTIs, the model of β thalassaemia major has been researched extensively to understand the degree of safety offered by the mandated screening technologies. The thalInd database is a repository of India thalassaemic information (Sinha et al., 2011). In India 64 β-globin mutations have been reported with IVS1-5C being the most common followed by the 619 bp deletion (Panigrahi & Marwaha, 2007; Verma et al., 2011). The general incidence of thalassaemia trait in India varies between 3 and 17% (Shah et al., 2010). With 100 000 β-thalassaemia patients, and 9000 thalassaemic annual births it is estimated that in India about 2 million units of PRBC are required for thalalssemic needs (Marwaha, 2010). It has been suggested that in the near future β-thalassaemia and related disorders are likely to emerge as the category of genetic disease that will have the most widespread impact on public health and health resources in India (Weatherall, 2010)
TTI COMPLICATIONS IN THE TREATMENT OF THALASSAEMICS The International Classification of Diseases (ICD) is the World Health Organization’s (WHO’s) ‘standard diagnostic tool for epidemiology, health management and clinical purposes, and used to predict mortality and morbidity statistics’. The ICD reports indicate that globally blood-borne viral infections mainly hepatitis C and B are the second most common cause of death after heart failure in the general population. Worldwide thalassaemia patients exhibit 0·3–5·7% HBsAg and 4·4–85·4% HCV positivity. As very good solutions are currently available for iron chelation, the disease management has shifted to address hepatocellular carcinoma (HCC) through TTIs (Di Marco et al., 2010). Typically 15% of acute HCV infections found in first time donors, and 54% in repeat donors have been shown to resolve spontaneously. Of the remaining, 85% of the serology negative donors will remain a source of occult infection. Generally 54–86% of acute HCV cases progress to chronic hepatitis, 25% of chronic hepatitis cases progress to cirrhosis, and 1–4% lead to HCC (Mukhopadhya, 2008). HBV has a unique disease pattern, in that the chronic carriers remain infectious throughout their life, albeit intermittently (Fig. 1; Vermeulen et al., 2012). In thalassaemics, as iron overload affects heart and liver, HCV and HBV infections add another layer of complexity to liver functionality. Thalassaemia major or intermedia subjects have been reported to exhibit HCC at a young age of 45 years © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
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compared with 66 years in the control group (Di Marco et al., 2010). The rate of progression of HIV-1 disease is also known to be faster in iron overload conditions. The early acquisition of infection and rapid disease progress is also ascribed to compromised immune status in thalassaemics. Although the above details are specific for thalassaemics, endemic proportion of diabetes (5% prevalence in the 20–79 age group) and heart problems (12% national prevalence) indicates the less than optimal health status of the Indian population.
initial serology reactive units. Several blood banks use rapid tests as supplemental to screening tests, which a decade ago had similar sensitivity as the screening ELISA test. However, over the years much improvement has been made in advancing the sensitivity and specificity of the screening ELISA/CIA tests and consequently rapid tests have become comparatively less sensitive (Laperche, 2013). The use of less sensitive rapid tests as supplemental test has caused much confusion regarding the accuracy of reactive screening results.
EFFORTS TO LIMIT TTI THROUGH BLOOD SCREENING IN INDIA
HBV prevalence in blood donors and contributing factors
Through the establishment in 1987 of National AIDS Control Organization (NACO), the Drug Controller General, India (DCGI) is vested with the responsibility to approve licensing of blood and blood products in 1992, and in 2002 by formulating a National Blood Policy, efforts have been made to make the blood supply safer in India (Regulatory requirements of blood and /or its components including blood products, http://cdsco.nic.in/html/guideline.htm; NACO, 2007). Testing of donated blood for anti-HIV was mandated in India in 1989, for HBsAg in 1992 and for anti-HCV in 2001 (NACO, 2007). However, as detailed in this article effective surveillance system which is required for realistic predictions of prevalence for effective public health policy for measures to curtail increase in infection is still lacking. Of the about 2700 blood banks nation-wide, nearly 40% are in the government category, 35% in private and 25% in private charitable categories (Choudhury, 2011). Depending on the category of blood bank there is a wide range in the quality of serology assays. The private hospitals and private charitable hospitals choose either third or fourth generation enzyme-linked immunosorbent assays (ELISAs) or chemiluminescent immunoassays (CIAs) that meet their internal quality and affordability criterion. A majority of government hospitals are provided third generation ELISA serology assay reagents free of cost, but of variable quality. A number of blood banks that are resource and facilities constrained, and also have very few donations are permitted to use rapid tests to screen (NACO, 2012). Using reagents of diverse quality and heterogeneous sample groups, in India the prevalence in general population is reported to be 0·36% for HIV-1/2, 0·09–7·89% for HCV, 2·5–4% for HBV (NACO, 2008–2009; Mukhopadhya, 2008; Abraham, 2012). Amongst the blood donor population including both volunteer and family/replacement donors the HIV-1/2 prevalence is 0·17–0·3% (Malhotra et al., 2013; NACO, 2008–2009), 0·4% HCV and 1·2% HBV (NACO, 2008–2009; Abraham, 2012). Subject to regional life-style habits, religious beliefs and nonrecommended practices a wide range in prevalence numbers have been recorded. In practice not all blood banks test the initial reactives in two replicates with the same reagent (which in itself does not control for assay specificity), and even fewer labs test repeat reactives with a confirmatory supplemental second assay. In India the majority of blood banks discard the © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
On the basis of HBsAg prevalence, India is considered an intermediate HBV endemic country. At a prevalence of up to 4% in the general population, and a disease burden of >50 million, India has the second largest pool of HBV carriers in the world (Abraham, 2012). Additionally 80% of HCC in India is virus associated, with 36–43% ascribed to HBV and 30% to HCV (Abraham, 2012). In India blood screening for HBsAg as the only marker for HBV was implemented in 1992. HBsAg is a direct measure of the viral protein marker, and depending on the reagent this can be detected as early as 38 days in plasma (Kleinman et al., 2009; Fig. 1). Through the presence of a 1000- to 10 000-fold excess of non-DNA containing sub viral particles, there is a relatively high concentration of HBsAg, resulting in early detection (Gerlich et al., 2007). HBsAg being a viral protein is a transient marker lasting only 63 days of infection in acute infections, which is a drawback (Vermeulen et al., 2012). Despite HBsAg being a viral protein and the anticipated high specificity, using the most advanced reagents 56% of repeat reactives could not be confirmed by a second neutralisation reagent assay (Stramer et al., 2013b). The use of less sensitive HBsAg assays and HBsAg mutations that are not detected by the antibodies allow the infection to proceed to occult carrier stage (Stramer et al., 2011). Occult B infection (OBI) is characterised by the absence of HBsAg, presence of anti-HBc with or without the presence of anti-HBs, and intermittently detected extremely low HBV viral load (VL), and VL as low as 5 IU mL−1 DNA has been shown to cause TTIs (Allain et al., 2013). Many of the thalassaemia prevalent countries also have high HBV occult carrier population, and in India it is estimated at 19% (Abraham, 2012). In studies from Northern India up to 10% of the blood donors were anti-HBc positive with 0·15–1·5% of these being DNA positive and hence potentially infectious (Makroo et al., 2012; Sen & Arora, 2012). In a study from Southeastern India 30% of the HBsAg negative donors were anti-HBc positive, and of them 30% were HBV DNA positive (Panigrahi et al., 2010). In a 2013 study from the United States anti-HBc positive donors (29 909) were 10 times more prevalent than HBsAg positives (2703), and 2·5% (723) of OBI repeat reactives were HBV DNA positive (Stramer et al., 2013b). Anti-HBc screening is mandatory in the United States and reactive units are excluded. In India through the use of HBsAg non-reactive OBI units, the transfusion risk of infectious HBV units remains high. While the screening for anti-HBc would identify nearly all chronic carriers, Transfusion Medicine, 2014, 24, 79–88
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Fig. 1. Serum markers and disease status of HBV infection. Profile of markers in acute resolving hepatitis B infection (top); chronic hepatitis B infection (Bottom). (Courtesy, Prof. J. P. Allain, University of Cambridge, UK.)
in India it also would result in high discard rate and donor deferral. In a system that is already burdened with insufficient blood supply the discard of non-infectious OBI units would be difficult to accommodate. The prevalent HBV genotypes in India are predominantly D, followed by A, C and additional recombinant forms in the Eastern region (Ghosh et al., 2013). A few of the HBV genotypes such as genotypes D and A2 do not reach high VLs, remaining below detection limit, and result in increased possibilities of infectious unit transfusions (Allain et al., 2013). The information regarding genotypes could also be skewed, as low VL samples are a challenge to amplify for genotype analysis. In India donors with HBsAg reactivity are referred to the gastroenterology departments and associates in hospital-associated blood banks and stand-alone blood banks, respectively.
HCV prevalence in blood donors and contributing factors The typical blood screening assay for HCV is for detecting antiHCV antibodies, and was mandated in India in 2001. Antibodies remain in circulation for a very long time lasting years, even after viral remission, hence antibody reactivity results suggest either a current HCV infection, a past infection or false positivity. As the HCV antibodies can be detected only after 65–70 days of WP, HCV has the longest period of non-detection of infectious units (Kleinman et al., 2009). The delayed immune response means that the viral propagation proceeds unchecked and remains high during this interval. The third generation anti-HCV ELISA and CIA assays are known to have high false positivity. In a study from the United States only 21–34% of repeat reactives could be confirmed by a second reagent Western blot (WB) recombinant immunoblot assay (RIBA) (Stramer et al., 2013a). Amongst the Indian blood donors the initial reactivity as detailed below was Transfusion Medicine, 2014, 24, 79–88
reported to be between 0·2 and 0·77%. Of the 0·2% (140) initial reactives, 63% (87) were non-reactive by a second ELISA reagent, and all of these were negative by confirmatory RIBA and for RNA (Tulsiani et al., 2012). In another study the initial reactive rate was 0·77%, and only 70% tested positive by a second assay and 21% positive by confirmatory immunoblot assay (Raghuraman et al., 2003b). Combination assays of HCV antibody and core protein viral antigen which can reduce the WP by 21 days are not widely used because of cost (Laperche et al., 2005). The availability of the confirmatory RIBA WB has been inconsistent and currently discontinued (Stramer et al., 2013a). In summary, >30% of the initial reactive samples are not confirmed with a second reagent, and of the remaining positives 50 transfusions resulted in 57% HCV prevalence. Age dependent breakdown of the prevalence was not provided to determine pre- and post-anti-HCV screening. From Western India where up to 7·48% thalassaemia is observed, using supplemental confirmatory assay 25% HCV prevalence has been reported (Jain et al., 2012). In a 3 year (1993–1995) long-term study by Choudhury et al. (1998) anti-HCV prevalence of 23, 30·7 and 35·9%, was observed for each consecutive year. In contrast, a prevalence of 2% was reported in another study involving 200 thalassaemics, again with no increase in TTIs with increased transfusions (Vidja et al., 2011). In order to understand if the high HCV prevalence of up to 57% in thalassaemics was unique to India or if it was a phenomenon also applicable to other thalassaemia endemic countries, publications from a few other countries were reviewed. The information in Table 1 points to the high prevalence of HCV in thalassaemics in all countries. In all of the above studies a breakdown of pre- and postserology screening results has not been provided. Because of the unique profile of HCV with very long WP for antibody development it is critical to analyse the data for pre- and postserology screening periods to gauge the impact of serology screening. In the only study providing a breakdown, Shah et al. (2010) indicate that 15% (six) of the children born after serology screening initiation were HCV positive. Overall 45% HCV prevalence was observed in this study, with double infections of HCV–HIV; and one triple infection for HBV also. Although Transfusion Medicine, 2014, 24, 79–88
they did not perform a comparative donor testing, the high prevalence in thalassaemics attests to infectivity of serology negative units. Despite the wide variance the high prevalence of HCV has led to a consensus call in all the publications for improved pretransfusion screening, including the suggestion to adopt nucleic acid technology (NAT) testing.
HIV positivity in thalassaemics in India Implemented in India in 1989, at 0·3% prevalence in blood donors HIV-1/2 has the lowest prevalence of the three viruses. In comparison a 2% HIV prevalence in thalassaemics of ages 8, 11 and 12 years was observed, suggesting TTI through serology qualified units (Shah et al., 2010). All three patients were coinfected with HCV, again pointing to transfusion as the cause. In a 200 patient one time study with a minimum transfusion of 10 units of blood, six (3%) HIV infections were observed in the 0–20 age group, again with all of them from postserology screening period (Vidja et al., 2011). Similarly in a 3-year annually monitored study of 39 patients of 3·5–9 years; one instance (2·5%) of HIV was observed (Choudhury et al., 1998). In a study with 126 patients of the age group 10 months to 22 years 4% HIV prevalence was observed. Although the study does not provide a breakdown of pre- and post-anti-HIV screening incidences, it is likely that a majority of the patients received serology negative units (Mathur et al., 2008). In another study with 96 patients for 2 years monitored at 6-month intervals a 1% prevalence of HIV was observed (Jain et al., 2012). Of the three viruses through early implementation it is possible to evaluate the impact of serology screening for HIV. The consensus of 1–4% prevalence in thalassaemics versus the observed 0·3% in donors points to the failure of serology assays to adequately interdict infectious HIV units. Through repeat transfusions, thalassaemic recipients indicate vulnerability to heterogeneous strains, resulting in recombinations and mutations of HIV virus.
NEED FOR NAT TESTING In spite of the mandated serology assays in India the reports for all three viral TTIs indicate the inadequacy of serology-only screening in ensuring blood safety. The abundant literature for post-serology infections for HIV and HBV and limited information for HCV definitely point to the necessity of rethinking about the sufficiency of mandated serology tests alone. Indeed a majority of the publications recommend the use of NAT testing. By targeting the very early event in viral propagation through the detection of viral genomes which are the source of infection, NAT tests provide the shortest WP. The most sensitive NAT tests amplify the nucleic acid several million folds to detect the presence of very low number of copies of viral NA. The current technologies are so fine-tuned for NA isolation, amplification and detection that the WP from the day of presence of virus in blood to the day of detection depends on the virus © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
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Table 1. Prevalence of anti-HCV serology marker in general population, blood donors and thalassaemics
Country
Serology testing since
% HCV prevalence General population
India
2001
1–1·9 (Sievert et al., 2011)
Iran Pakistan
1996 2003
Malaysia
1995
0·3–1 (Azarkeivan et al., 2012) 4·7 (Waheed et al., 2009; Sievert et al., 2011) 1·5–2·3 (Duraiswamy et al., 1993)
Thailand Egypt
1991 1999
2·8 (Sievert et al., 2011) 15 (Sievert et al., 2011)
Blood donors
Thalassaemics
0·4 (NACO, 2008–2009; Abraham, 2012) 0·12 (Azarkeivan et al., 2012) 0·5–8·9 (Waheed et al., 2009; Sievert et al., 2011) 0·4 (Haslina et al., 2012)
16–26 (Mukhopadhya, 2008; Abraham, 2012) 19 (Di Marco et al., 2010) 48·6 (Waheed et al., 2009; Di Marco et al., 2010) 13–22 (Lee et al., 2005; Di Marco et al., 2010) 21·2 (Di Marco et al., 2010) 24–76 (El-Faramawy et al., 2012; El-Shanshory et al., 2013)
0·31–3·54 (Sievert et al., 2011) 8–14·7 (Sievert et al., 2011; El-Shanshory et al., 2013)
doubling time. Assuming that infectivity first occurs with one viral copy in 20 mL plasma (mL of plasma per unit of PRBC), based on results from seroconversion panels infectious WP days have been calculated (Kleinman et al., 2009). When practiced in the highly sensitive individual donation (ID) NAT testing format, with an analytical sensitivity for HCV at eight copies per millilitre (1 IU = 2·5 copies), with a doubling time of 0·45 days, the infectious unit is detected within three WP days, reducing the infectious risk period by 58 days from third generation ELISA anti-HCV serology testing. To date transfusion of several million units of blood qualified by ID-NAT testing has not resulted in a single case of HCV transmission. For HBV through ID-NAT testing format, with an analytical sensitivity of 12 copies per millilitre (1 IU = 5·6 copies), with a 2·5-day doubling period of VL, the infectious unit is detected within 15 WP days, reducing the infectious risk period by 21 days over HBsAg serology testing. To date for several million units tested by ID-NAT there is only one instance of TTI caused by HBsAg negative WP unit with 1·5 copies per millilitre of HBV DNA (Vermeulen et al., 2012). Globally, because OBI serology marker screening is impractical, OBI transmissions can be interdicted only through sensitive NAT screening. ID-NAT testing for HIV-1, with an analytical sensitivity of 16 copies per millilitre (1 IU = 0·6 copies), with a doubling time of 0.85 days detects infectious unit within 6 WP days, reducing the infectious risk WP by 13 days over anti-HIV serology testing. To date the release of millions of ID-NAT tested units has resulted only in a single case of HIV transmission with 3–4·5 Cps mL−1 (Salles et al., 2013). The single case each of HBV and HIV, are reminders that while ID-NAT testing offers the maximum safety, it still does not provide risk-free blood. ID-NAT is the most sensitive format because each sample is tested individually, and directly reflects the analytical sensitivity of the assay. A second method of practice is the pooled testing which introduces an element of dilution, with decrease in analytical sensitivity proportional to the pool size. This is also referred to as screening sensitivity, and also results in decreased clinical sensitivity. In the past a number of countries practiced pooled testing format for a variety of reasons including: (i) very limited option of automation for high through-put required © 2014 The Author Transfusion Medicine © 2014 British Blood Transfusion Society
for individual unit testing, (ii) high cost for each result, (iii) challenges in implementing complex technology, (iv) concerns of amplicon contamination through NA amplification of a large number of samples, (v) insufficient proof of the benefit of IDNAT, (vi) low prevalence in advanced countries coupled with (vii) superior quality and additional serology assays resulting in insignificant cost-benefit. Currently, these factors have been addressed through the possibility of high-throughput screening and automation, resulting in ease of implementation, and closed systems to control contamination. Most importantly, experience with NAT testing in the past 15 years has led to important revelations about the need for ID-NAT in moderate prevalence countries. Several retrospective studies have demonstrated the correlation of ID-NAT with detection of viruses in the previously missed pooled units, and the estimates of TTIs that could have been interdicted by ID-NAT (Shyamala, 2014). All these factors have led a large number of countries to adopt ID-NAT testing. Additionally, HBV DNA screening as an indicator of infectivity is more effective in countries with high to medium endemicity and where anti-HBc is not mandated. Currently in India 7% of the 7·9 million units of annually donated blood is screened by ID-NAT testing. The results from a multicenter study indicated serology negative NAT reactive yields to be in the range of 1 per 1333 (nine yields for 12 000 units) with six of nine yields being HBV (Makroo et al., 2008). Being a multicenter study the serology tests used to define NAT yields were a mix of third and fourth generation assays from a variety of vendors. In another study with 73 898 tests with advanced serology tests, a NAT yield of 1 per 610 donations was observed; with 60% being HBV, 30% being HCV, 0.8% being HIV-1, and 8% being double infection HBV and HCV (Agarwal et al., 2013). Using this same donor base and an improved HBV detecting NAT assay HBV NAT yield of 1 per 301 was reported (Chatterjee, 2012). The improved HBV reagent detected higher percentage of OBI cases, with >80% having very low VL of 62% being HBV yields. Nearly 70% of the HBV NAT yields are OBI cases, which being HBsAg negative are currently being transfused as ‘safe blood’. While, whole blood, particularly fresh whole blood (stored for less than a week) remains the product of choice for emergency bleedings, the primary transfusion need of thalassaemics is for PRBCs. However, in India only 30% of the blood banks have component preparation facilities (Marwaha, 2010). As a result, at many facilities, thalassaemics and general recipients irrespective of their specific need for PRBC or platelets are provided with whole blood. While there are several immunohematology reasons to discontinue this practice, there are also compelling TTI reasons to discontinue this practice. Nearly all of the HIV1, HCV and HBV VL in the blood is present in the plasma fraction. It is estimated that one unit of PRBC has 20–30 mL of plasma, and one unit of platelets has 50 mL of plasma versus a unit of whole blood which contains 180–220 mL plasma. Hence the whole blood transfusion results in six to seven fold excess over PRBC. The total VL transfused has been shown to be directly correlated to cases of TTI (Kleinman et al., 2009; Allain et al., 2013). There are numerous reports of TTIs with plasma transfusion, while PRBC and platelets from the same unit have not resulted in TTI. Hence in all situations TTIs can be minimised through the required use of components.
CONCLUSION The true value of diagnostics depends on assays that are early predictors of infection offering maximum blood safety. However, through breach in ‘safe blood transfusion’ thalassaemics are confronted by new and more severe health challenges of TTIs contributing to increased mortality, and this can be extrapolated to general recipients as well. It can be undoubtedly stated that through the current serology screening practices, TTIs for HIV-1, HCV and HBV are proceeding unchecked. Despite official positions, the approach of exclusive volunteer non-remunerated safe donors and vigilant donor deferral is not an immediately implementable way to curtail this
REFERENCES Abraham, P. (2012) Viral hepatitis in India. Clinical Laboratory Medicine, 32, 159–174. Agarwal, N., Chatterjee, K., Coshic, P. & Borgohain, M. (2013) Nucleic acid testing for blood banks: an experience from a tertiary care centre in New Delhi, India. Transfusion and Apheresis Science, 49, 482–484. Allain, J.P., Mihaljevic, I., Gonzalez-Fraile, M.I. et al. (2013) Infectivity of blood
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increase. From a blood supply point of view, family donors are a motivated population that significantly contributes to the Indian blood supply and cannot be deferred without due consideration of the evidence. Transforming the donor base to voluntary repeat donors is a sociological change which will take a long time to accomplish. For the immediate, implementable testing practice for maximum safety is the solution. Although the argument of unaffordability of ID-NAT testing has been the premise for pooled testing, the residual risk of the resulting TTIs places a serious burden on the health care, finances, society and human life. True health care costs applicable to Indian scenario have not been available. The cost of advanced stage HIV-1 treatment in Thailand has been projected at USD $1158 (van Hulst et al., 2009). As emphasized in the review by Shyamala (2014), in India the cost is substantially borne by the government and this expense could be decreased by improved blood screening to prevent potential TTIs. The situation for hepatitis is even grimmer, as the clinical symptoms of the infection take a long time to manifest and the magnitude of the disease burden will not be apparent for years to come. Additionally, unlike for HIV, there is no national policy for patient treatment for hepatitis. The economic burden of HBV/HCV infection is substantial even in the early stages of infection, and escalates steeply with advanced stages of infection. The annual cost of weekly injectable pegylated interferon therapy available for hepatitis is billed at INR 884 000 (USD $15 000). The treatment costs for HCC and decompensated cirrhosis in Thailand are estimated at USD $6760 and USD $13 973, respectively; these costs are the highest in the health care management (van Hulst et al., 2009). These observations are a wake-up call for providing additional layer of security to national blood supply through NAT testing.
ACKNOWLEDGMENT The concept, literature review and writing was solely by the author. The author likes to thank Mr. Sangeet Kini for providing valuable background information.
CONFLICT OF INTEREST V. S. is a part-time consultant for Novartis Diagnostics.
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