Diagnostic accuracy of blood centers in the screening of blood donors for viral markers

Introduction Blood transfusion still remains a life saving intervention in almost all healthcare facilities worldwide. Screening of blood donors/blood units is done in almost every blood bank facility before the blood units/blood components are transfused to prevent transfusion-transmissible infections. The kind of testing kits or the methods used by a facility and the technical expertise of the personnel greatly affects the screening results of a facility. This study was aimed at evaluating the diagnostic accuracy of five hospital-based blood bank testing facilities (Komfo Anokye Teaching Hospital KNUST, Kwame Nkrumah University of Science and Technology, Agogo, Bekwai and Sunyani) that used rapid immunochromatograhic assays (RIA) in screening blood donors/blood units in Ghana. Methods Blood samples (300) from the five testing facilities and their screening results for hepatitis B surface antigen (HBsAg), antibodies to hepatitis C virus (HCV) and human immunodeficiency virus (HIV) using RIAs were obtained. All the samples were then analysed for the three viral markers using 3rd generational enzyme linked immunosorbent assay (ELISA) kit as the gold standard. Results The mean false positive for HBsAg was 2.2% with Bekwai testing facility having the highest of 4.4%. For HCV, the mean false positive was 2.8% with Agogo and Bekwai testing facilities having the highest of 8.7% respectively. For HIV screening, the mean false positive was 11.1% with Bekwai testing facility having the highest of 28.0%. The mean false negative for the facilities were 3.0% for HBV, 75.0% for HCV and 0.0% for HIV with KATH having the highest of 6.3% for HBV, Bekwai having the highest of 100% for HCV and no facility showing false negative for HIV. Mean sensitivity of the screening procedure for the facilities was 97.0%, 25.0% and 100.0% whilst the mean specificity was 97.8%, 97.2% and 88.9% for HBV, HCV and HIV respectively. Statistical comparison among the testing facilities showed no significant differences among the various testing centres for HBV screening; however, significant differences were obtained for HCV and HIV screening. Conclusion This study has shown that there is no standardised screening procedure for blood bank testing facilities in the country. There is therefore an urgent need for an internal and external control body to oversee screening procedures in blood banks across the country.


Introduction
Transfusion transmitted infections (TTIs) still remain major public health problem encountered by the health delivery systems in many developing countries mainly due to under resourced facilities and lack of requisite staff [1]. Blood transmitted infections involving pathogenic viruses are the most prominent in transfusion medicine [2]. In spite of all the scientific advancements and improvements in technology aimed at improving the safety of blood donation, viruses and bacteria mainly hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) and syphilis still remain the most transmitted infectious pathogenic agents passed on from donor blood to recipient through transfusion [3]. In 2001, the World Health Organisation (WHO) estimated that transfusion of unsafe blood accounted for 8 -16 million hepatitis B virus infections, 2.3 -4.7 million hepatitis C infection, and 80,000 -160,000 human immunodeficiency virus (HIV) infections each year [4]. Even though there are routine tests for these viruses, the greatest risk are donations given in the infectious window period (WP), which is the time between development of infectious viraemia and reactivity by routine serological or nucleic acid technology (NAT) donor screening tests [5]. Blood donors like anyone else, could occasionally carry an infectious agent, sometimes for a long period without having any clinical signs or symptoms [6] thus possessing threat to future recipients through the blood products from such donors. Several screening tests/assays have been developed over the years to overcome this threat; Some of these assay techniques used in screening blood/blood products prior to transfusion include immunochromatographic assay, enzyme-linked immunosorbent assay (ELISA) and nucleic acid test (NAT) or polymerase chain reaction (PCR) assay techniques. The effectiveness of all these tests in interdicting contaminated units of blood/blood products depends in part on the point in time when an infected donor provides the unit relative to the individual's exposure to the virus [7], adequate training of laboratory staff on risks of transfusion-associated viral transmission and also their ability to follow standardised procedures in applying all these testing technologies as well as adhering to good quality control measures so as to prevent testing/procedural errors [5,8,9].
Issues concerning blood safety are mostly in two parts: how to identify infectious donor or blood unit and thus prevent its onward transfusion in so doing safeguarding the recipient; and also how to prevent false positives which result in deferment of potential blood donors or discarding of donated blood. The success or otherwise of such an action depends on two main policies. The first strategy is the adaptation of a national transfusion policy for the selection of blood donors which mainly aims at excluding blood donors with higher risk for infections like HIV, HBV, HCV and syphilis. The second is the application of assay technologies with high specificity and sensitivity so as to be able to identify all true positive individuals/blood units and true negative individuals/blood units [10]. Screening of blood donors is a critical issue as the outcome of the test if not properly performed can result in serious consequences for either the blood service or the blood donor. False positive result can lead to a larger number of blood donors being deferred, while a false negative testing may jeopardize blood safety [11]. Improperly trained laboratory personnel may also produce a false screening result. Transfusion of an infected blood to an individual is a crime [12] and to avoid this, strict haemovigilance and quality control systems (both internal and external) need to be put in place in all blood centre facilities. Currently in Ghana, the policy of blood donor selection include or exclude a donor on the basis of a health check questionnaire and a visual physical examination together with a mandatory screening for HIV, HBV, HCV and syphilis [13]. But in Ghana and most sub-Saharan Africa countries, resources for viral marker screening among blood donor are limited, with the exception of immunodeficiency virus screening which is mostly supported by long-term foreign aid [14]. Antibodies to hepatitis C and hepatitis B surface antigen (HBsAg) even though routinely done do not enjoy such attention and supervision hence the type of assay technology and brand of test kit used in a blood facility is the choice solely of the individual facilities. Most facilities currently use lateral flow immunochromatographic rapid tests or enzyme immunoassays (EIA) test kits in the screening of blood donors for viral markers [13,15]. Currently there is limited information/data on the evaluation of the residual risk of viral transmission in Ghana, but Allain et al (2003) observed that neither the dipstick assay nor latex agglutination assays currently in use for HBsAg screening have sufficient sensitivity in detecting HBV in infected blood, with latex agglutination and dipstick methods presenting a false negative rate of 46% and 29% respectively. The primary objective of this study therefore was to retest samples from five testing facilities who used rapid immunochromatographic assay to screen blood donors with enzyme linked immunosorbent assay (ELISA), which is the method recommended by WHO for all blood banks [16] and compare the results from the various facilities with ELISA results.

Reactivity of the viral markers at the various testing
facilities upon re-testing with ELISA Table 1, 300 samples were analysed during the study, of which 100 came from KATH and 50 each from KNUST, Bekwai, Agogo and Sunyani blood banks respectively. When the samples were retested with ELISA, 6 (2.2%) were false positives and 1

Specificity and sensitivity of rapid immunochromatographic assays (RIAs)
The specificity of a testing facility is defined as the proportion of population/subjects without an infection that the testing facility will truly confirm negative for the infection being tested whilst the sensitivity of a testing facility is the defined as the proportion of population/subjects with an infection that the testing facility will truly confirm positive for the infection being tested [17]. The specificity and sensitivity for each of the testing facility were also calculated as shown in Bekwai had the lowest (0.0%) for HCV with all the centres having 100% sensitivity for HIV.

The youden index for acceptability of testing methods
The Youden index also known as the J-index [18][19] was used to assess the accuracy or the acceptability of assaying results from each of the testing facilities. The higher the J-index of a facility, the better is the accuracy of the result from that facility as well as the more acceptable the result. J-index has an upper limit of 1.0.
From Table 2, the average calculated J-index for HBV assaying at the various facilities was 1.0, with KATH having a value 0.9 which is lower than the average. The calculated average J-index for HCV screening at the various facilities was 0.2 with KATH having a value of 0.5 which is far above the facility average whilst Bekwai had a value of -0.1 which was much lower than the facility average. For HIV testing at the various facilities, the average J-index was 0.9 with Bekwai testing facility having value of negative 0,5.

Positive Predictive Value (PPV)
PPV defines the proportion of individuals with a positive test result who do actually have the disease/infection [17]. PPV is calculated by using the formula ( TP/ (TP+FP)'>TP/ (TP+FP)) and it gives the showed the same level of NPV of 25.0% ( Table 2).

Positive Likelihood Ratio (LR+)
The odds ratio that a positive test result will be observed in an infected population compared to the odds that the same result will be observed among a non-infected population also known as the positive likelihood ratio was calculated for each of the testing facilities [20]. Agogo facility had the highest (43.0) positive likelihood ratio for HBV testing, whilst Bekwai facility had the lowest rate of 22.5 ( Table 2). For HCV testing, Bekwai facility had the lowest rate of 0.0 whilst Agogo had the highest rate of 2.9 ( Table   2). For HIV testing, KATH facility showed the highest positive Likelihood ratio of 16.5 whilst Bekwai facility had the lowest rate of 0.0 ( Table 2).

Negative Likelihood Ratio (LR-)
The odds ratio that a negative test result will be observed in an infected population compared to the odds that the same result will be observed among a non-infected population also known as the negative likelihood ratio (LR-) [20], was determined for each of the

Bland-Altman estimation
The Bland-Altman analysis [22] was used to compare the testing facilities based on their testing bias or degree of estimation in using RIAs rather than ELISA in testing for the presence of the three viral markers. As shown in

Discussion
Safe transfusion practice has been a matter of concern for the health care providers since the commencement of blood transfusion practices and the subsequent discovery of blood transmissible infectious agents [23]. To ensure safe transfusion practices across the world, the World Health Organisation (WHO) declared 2012 as the year for attaining 100% testing for infectious viral markers in blood products [24]. The status of this target in most developing countries especially Sub-Saharan Africa is however unknown presently as the region is burdened with a lot of social and scientific factors that works against safe transfusion practices [24,25].  acquiring an infection through blood or a blood being rejected for blood donation is higher in some facilities than others and there is still more work to be done if all our testing facilities are to be at par.
Even though RIAs are useful screening methods in blood bank testing facilities in low-resource settings however, the method should be carefully validated locally before being used for screening as their performance may varies by location.

Conclusion
The evaluation of the performance of five hospital based testing facilities in the use of rapid immunochromatograhic assays to screen blood donors for HBV, HCV and HIV in Ghana showed that the strategy being used by the facilities now is inadequate to prevent transmission of HBV, HCV and HIV through blood transfusion in Ghana due to high residual risk of transmission of these viruses through blood transfusion. There is thus an urgent need for an effective and efficient control system to be put in place for screening of blood donors at these and other facilities throughout the country.

Competing interests
The authors declare no competing interests.

Authors' contributions
Elliot Eli Dogbe and Fareed Arthur conceptualized and designed the study. Elliot Eli Dogbe performed the laboratory work and also performed the statistical analysis of the data. All the authors analyzed the data and contributed to the drafting of this manuscript.
Also, they have read and approved the final version.

Acknowledgments
The authors wish to thank the staff of the various blood banks for their advice and assistance in the recruitment of the study participants. The findings, interpretations and conclusions expressed in this research findings are entirely those of the authors and do not necessarily represent the views of the institutions where they work for or are affiliated with.