Fourth-Generation ELISA Screening of Blood Donors: A Reliable Alternative to Nucleic Acid Testing

The gold standard for HIV screening of blood donors is individual nucleic acid amplification testing (NAT). However, individual NAT testing is cost-prohibitive, especially in a resource-limited setting. The fourth-generation ELISA that detects both p24 antigen and antibody to HIV-1 and 2 has been recommended as the minimum test for HIV to enhance blood transfusion safety and can be an alternative to NAT testing in resource-limited settings. The aim was to assess the performance of a fourth-generation ELISA in use at a regional blood transfusion service using nucleic acid amplification testing on units of screened blood negative to HIV. The study was a cross-sectional study conducted at the National Blood Transfusion Service center and the Plateau State Virology Research Centre, both in Jos, Nigeria. Between August and October 2016, one thousand and eighteen voluntary blood donors were recruited consecutively and had their samples tested using fourth-generation ELISA. One thousand p24 antigen-negative samples were pooled for NAT in an aliquot of 50 samples. All the pools of fifty samples of 1,000 HIV p24 antigen-antibody negative donor blood screened by the fourth-generation ELISA tested negative for HIV RNA on nucleic acid amplification. The yield of pooled NAT for HIV after a fourth-generation ELISA screening of blood donors was found to be zero in this study, thus establishing the fourth-generation ELISA's reliability. Therefore, we recommend adopting the fourth-generation ELISA test as a minimum requirement for blood donor screening.


INTRODUCTION
lood transfusion was a significant route of B 1 transmission of HIV in the past. As a result of the inoculums' size, transfusion of infected blood is associated with an almost 100% chance of successfully 1 transmitting the virus. However, the introduction of mandatory pre-transfusion screening for Transfusion Transmissible Infections (TTIs) led to a decline in the transmission of HIV through this highly efficient 2,3 route.

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The development of sensitive assays against the HIV p24 antigen and nucleic acids significantly reduces the diagnostic window, permitting earlier detection of the 4,5 virus The tests based on the detection of viral nucleic acid have been used to identify HIV in pools of blood 6 samples. With meticulous donor selection, nucleic acidbased testing can further significantly reduce the chances 4,5 of window period donations and recipients' inoculation. However, running NAT on individual donor samples is expensive, hence not done in most developing nations' 7 blood banks. Although fourth-generation ELISA is not a substitute for individual NAT, it can be an alternative, cost-effective method for improving blood safety in 8 developing countries. This study, therefore, seeks to establish the reliability of the fourth-generation ELISA in use at the North-Central regional National Blood Transfusion Service (NBTS).

Background of the study area
The work was carried out at the National Blood Transfusion Service (NBTS) and the Plateau State Human Virology Research Centre (PLASVIREC), Robert Gallo House, Jos, Nigeria. The PLASVIREC is a research Center with active collaborations with the USA Centers for Disease Control and Prevention and the Institute of Human Virology of Nigeria. The Center conducts molecular testing of viruses, among which are the HIV and hepatitis viruses.

Study design
The study was cross-sectional. Study subjects were blood donors aged 18-65 years recruited during routine donor drives organized by the NBTS. Relevant data were obtained from the subjects with the aid of a selfadministered and researcher-administered questionnaire for literate and uneducated subjects, respectively. The questionnaire addressed the socio-demography of the participants and risk factors for TTIs. Prospective blood donors who consented and passed the donor selection criteria were recruited consecutively.

Sample collection and analysis
Three-Stage multi-stage sampling was used: all prospective donors who consented to participate in the study were enrolled. After that, selected participants were weighed, and finger-prick blood samples obtained ® for haemoglobin estimation using the HemoCue haematology point of care device. Those whose weight and haemoglobin concentration were within acceptable limits were selected and given a light refreshment, after which venous blood was collected in adult blood bags. Subsequently, 5 ml of blood was expressed from each blood bag tubing into plain vacutainers. The samples obtained were transported in ice packs to the NBTS blood bank, where they were centrifuged, and the sera tested for HIV using a fourth-generation ELISA  The batch was valid when no flag appears for any of the controls. We adhered strictly to the manufacturer's protocol.

Data Analysis
The data obtained were analyzed using SPSS Statistics for windows version 16  The cycle threshold (Ct) for HIV-1 is above the limit of the assay, or no cycle threshold value for HIV-1 is obtained. Report result as "HIV-1 RNA not detected". <2.00E+01 copies/ml Calculated copies/ml is below the Limit of Detection of the assay. Report result as "HIV-1 RNA detected, less than 20 HIV -1 RNA copies/ml". <2.00E+01 copies/ml and -1.00E+07 copies/ml Calculated results are ≥ 20 copies/ml, and less than or equal to 1.00E +07 copies/ml are within the Linear Range of the assay. >1.00E+07 copies/ml Calculated copies/ml is above the Linear Range of the assay. Report result as "greater than 1.00E+07 copies/ml". Chi-Square with Fisher's exact test were used to determine associations between categorical variables. A p-value of < 0.05 was considered statistically significant.
The results obtained were presented in tables.

Ethical consideration
The study was a part of a larger study titled "The Outcome of Nucleic Acid Amplification Test in HIV p24 Antigen-Negative Blood Donors in Jos, Nigeria." The study was undertaken after due approval from the NBTS' Ethical Committee (REF. No: NBTS/EC/EA/44).

RESULTS
The mean (±SD) and participants' median age were 29.7 ± 10.4 and 26 years, respectively. The youngest donor was 18 years, while the oldest was 65 years. There were more males (69.5%) than females (30.5%) with a male: female of 2.3:1 (  Table 3

DISCUSSION
This study assessed the performance of a fourth-TM generation (Bio-Rad Genscreen ) assay in use at a regional NBTS by subjecting ELISA-negative samples to nucleic acid amplification testing. Of the one thousand fourth-generation ELISA-negative p24 samples, none was reactive when subjected to Realtime PCR. This finding is similar to that reported by 10 11 Chigurupati Based on the outcome of pooled NAT in this study, none of the blood donors was found to have a second diagnostic window-a period that coincides with the absence of specific antibodies and a decline of p24 antigen below the detection limit of the fourth-generation ELISA. Our finding is at variance with reports by others who found a second diagnostic window with the fourth-generation 14,15 ELISA. The zero yields of NAT in this study may result from the category of blood donors recruited. All the subjects in this study were voluntary non-remunerated blood donors considered the safest category of blood donors. More often than not, voluntary blood donors seem sure of themselves regarding TTI's, and in particular, HIV. The absence of NAT yield in this study may also be due to the method used: pooled specimen as against individual NAT. This was possible because of the potential for progressive dilution of a positive sample with increasing 9 pool size. Westreich et al. reported that an increase in pool size from 16 to 100 reduced the likelihood of HIV detection by 15 to 30%. Choosing a NAT with a lower limit of detection of <20 viral copies/ml, such as the one used in carrying out this research, may minimize the effect of increasing pool sizes on the pooling algorithm 9 16 sensitivity. The fourth-generation ELISA's performance in this study shows that it can be relied upon for blood donor screening in our setting, providing a cost-effective alternative to NAT.

CONCLUSION
Our study found a zero yield of pooled NAT for HIV after fourth-generation ELISA screening of blood donors. This finding was in agreement with other studies, thus confirming the fourth-generation ELISA's reliability for blood donor screening in our environment. We recommend adopting the fourth-generation ELISA as the minimum test for TTIs; however, additional tests for blood safety are advocated.

Limitations
Due to the high cost of running individual NAT on donated blood units, pooled NAT was used to assess the Fourth-generation ELISA's reliability in this study.