Implementing fourth generation human immunodeficiency virus enzyme-linked immunosorbent assay: One step forward in blood safety

Blood transfusion is a life-saving procedure and at the same time it always carries some inherent risks. In the context of the 20^th century, with the rapid advancement in the diagnostics and laboratory medicine the early detection of markers for transfusion transmitted infections (TTIs) have been reduced from several weeks to few days. This has diminished the residual risks of TTIs to as low as 1 in 677,000 units for human immunodeficiency virus (HIV), 1 in 103,000 for hepatitis C virus (HCV) and 1 in 63,000 for hepatitis B virus (HBV).[1] At present, the greatest threat to the safety of blood supply is the donation of blood by seronegative donors during the infectious window period. Such people represent new or incident infections during pre sero-conversion period.

Blood transfusion in the 21^st century is as safe as ever, with the implementation of nucleic acid testing (NAT) almost zero risk transfusion is possible if at all it is not challenged by the newer emerging pathogens. Although the NAT screening reduces the window period of viral infection, in India the conventional 3^rd generation enzyme linked immunosorbent assay (ELISA) still remains the most common screening test for TTIs. The overall cost and infrastructure development are the major challenges for its universal implementation. So, an ideal screening test in the resource constrained set-up should be highly sensitive, easily operable and cost-effective as mentioned by Malhotra et al.[2] It also worth mentioning that an accurate estimates of the risks of transfusion-transmitted infectious diseases are essential for monitoring the safety of the blood supply and evaluating the potential effect of new screening tests.

The most direct way of estimating the risk associated with transfusion is to study the rate of infection prospectively in transfusion recipients. However, such studies are to be extensive, may be almost impossible considering the low prevalence of TTI markers[3] and in the absence of a well-organized hemovigilance network in India. There are only a few current estimates of the residual risks of TTIs by blood transfusion of HBV or HCV and HIV in India.[4] However, considering the significance of infective window period, introduction the 4^th generation ELISA in HIV screening will definitely escalate the possibility of detecting HIV sero-conversion earlier in blood donors especially in countries where the prevalence of HIV is on the relatively higher side. This is due to the detection of HIV core protein (p24 antigen [Ag]) that appears transiently in blood donors prior to seroconversion. As per the World Health Organization recommendation for HIV testing, in addition to the detection of antibody, the screening assay should preferably also employ the detection of Ag. It further reduces the serologic window period by 3-7 days.[5]

In a very recent published comparative analysis on the performance of 3^rd versus 4^th generation HIV ELISA in North India showed, increased sero-reactivity is detectable by 4^th generation ELISA. On analysis of 1075 donors’ sample, an additional 4 samples were reactive by 4^th generation ELISA with one of them was confirmed positive by Western Blot (WB) analysis.[6] The present status of WB as confirmatory test does have limitations and drawbacks due to non-specific band reactivity, contamination with human cellular Ag and positional presence of antigenic bands on WB strip. All these may relate to the indeterminate results, which may vary from none in high prevalence populations to 50% or more among blood donors in ELISA reactive samples.[7]

In the study by Malhotra et al.,[2] in the current issue of the journal showed 4^th generation ELISA could detect higher number of seroreactive samples (37 vs. 14/ 10,200 donations), but the difference in seroprevalence expressed per 1,000 donations was not statistically significant. The difference in the seroprevalence of HIV among blood donors in various groups and subgroups using 3^rd and 4^th generation ELISA was also non-significant. One of the observation worth mentioning from this study was the 4^th generation ELISA detected all the 11 samples, which were reactive to both WB and 3^rd generation ELISA with an additional yield of 0.58 window period units per 1,000 donations (6 WB confirmed reactivity out of 10,200). However, a high rate of false reactivity was also observed (20 out of 37) in 4^th generation ELISA against WB assay. This could be an explanation to the present status of WB as a confirmatory test for HIV as mentioned previously.

Since, NAT was not done to confirm the results of the 4^th generation ELISA due to financial constraints, but there are few similar studies from the developing countries which had estimated the addition of p24 Ag, minipool NAT, and individual donation NAT assays would detect 3.9, 8.3 and 10.8 window period units per 10,00,000 in first-time donors respectively.[8]

Inclusion of the 4^th generation HIV ELISA in the national blood screening mandate is likely to improve the safety of blood components without much financial burden. However, more multi-centric studies are required across the country to evaluate its effective potential. Implementing improved blood screening along with better donor education and identifying low-risk donor population are the major pillars of blood safety in the developing world, simply a highly sensitive blood donor screening strategy may not be adequate.