Impact of nucleic acid testing in double screening of blood donations in Northern India: A single-center experience

INTRODUCTION

India has an annual requirement of 12 million units for transfusion.^[1] Due to the high prevalence of infections in the donor and general population in India, especially with respect to the three major transfusion-transmitted infections (TTIs) of viral origin, namely, hepatitis B virus (HBV) hepatitis C virus (HCV) and human immunodeficiency virus (HIV),^[2] serological screening is mandatory for the three viral TTIs in blood banks in India.^[3] Even though the use of advanced serological techniques improves the sensitivity of detection of TTIs, complete reliance on serology-only screening techniques still leads to missing of some of the donations harboring infections.^[4,5] The conventional serological screening assays have wide “window period” of detection and may also miss occult hepatitis B infections (OBI), immune-silent carriers, and immunologically variant viruses.^[6] Consequently, the Drugs and Cosmetics Act and the National AIDS Control Organization (NACO) allow for the introduction of better techniques to improve blood safety.^[7,8]

The national seroprevalence of hepatitis B and hepatitis C is 0.95% (0.89–1.01) and 0.32% (0.28–0.36), respectively.^[9] Moreover, the distribution of the disease burden among the states is unequal. The seroprevalence of HCV in Punjab has been found to be about 3.29% in the general population.^[10] Punjab, at 1.35%, also has the highest seropositivity for hepatitis C among blood donors, much higher than the national average of 0.34%. In fact, unlike the rest of the country, the seropositivity of hepatitis C is higher than that of hepatitis B (0.65%) in blood donors in Punjab.^[11,12] The heightened prevalence of HCV in Punjab can be traced to a combination of factors including insecure medical practices, unsafe therapeutic injections and surgeries, intravenous drug use, blood transfusions, hazardous dental procedures, and risky sexual practices.^[13]

Molecular screening of HBV, HCV, and HIV by nucleic acid testing (NAT), in addition to serology, can improve the sensitivity of screening. It offers the potential of reduction of transmission of infections through transfusion.^[14] A donated blood sample in which serological assay failed to detect the infection, but NAT detects a viral genome is referred to as a NAT yield.^[15,16] The use of automated systems offers additional benefits of simplification of requirements of facilities in the blood center. The introduction of technological advances and automated molecular screening systems requires an investigation of their potential benefit.

This study reports the 6 year experience of implementing minipool NAT (MP-NAT) in a tertiary care hospital in North India. The data include the incremental yield obtained by employing a highly sensitive NAT in minipool format to serononreactive blood donations and subsequent quantitation of viral targets.

MATERIALS AND METHODS

Molecular screening

Only donations that showed non-reactivity to screening for all serology targets were further tested by NAT. NAT was performed using the cobas® TaqScreen MPX Test version 2.0 (MPX2; Roche Molecular Systems, Branchburg, NJ, USA) on the cobas s201 platform (Roche Instrument Center, Rotkreuz, Switzerland). The system includes an automated sample pooling on the Hamilton MICROLAB® STARlet IVD Pipettor, automated sample preparation on the cobas Ampli Prep (CAP) instrument, and automated amplification and detection using the cobas TaqMan (CTM) analyzer. The MPX2 is a multiplex, multidye test employing polymerase chain reaction (PCR), for the simultaneous detection and identification of HBV, HCV, and HIV through detection of the targets HBV deoxyribonucleic acid (DNA), HCV ribonucleic acid (RNA), HIV-1 group M, HIV-1 group O, and HIV-2RNA.

Screening was performed on minipools of 6 samples. Reactive minipools were resolved by individually testing all 6 units of the minipool to identify the reactive donations and the virus. NAT was performed following the manufacturer’s instructions for the assay on an automated platform. The screening algorithm is summarized in Figure 1. The 95% and 50% detection limits for MPX2 are shown in Table 1.^[17-20]

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Figure 1:

Flow chart of minipool-nucleic acid testing algorithm. HBV: Hepatitis B, HCV: Hepatitis C, HIV: Human Immuno deficiency Virus, NAT-PCR: Nucleic Acid Testing-Polymerase chain reaction, MP- NAT: Minipool-Nucleic Acid Testing

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Table 1: Detection limits of the molecular assays.

Target	cobas® TaqScreen MPX Test, v2.0donor screening assay		cobas® AmpliPrep/cobasTaqManviral load assays
	95% LOD*[16]	50% LOD*[17]	LLOQ[18,19]	95% LOD[18,19]
HIV-1 Group M*	50.3 IU/mL	9.4 IU/mL	-	-
HCV	6.8 IU/mL	1.3 IU/mL	15 IU/mL	11 IU/mL
HBV	2.3 IU/mL	0.46 IU/mL	20 IU/mL	8.2 IU/mL

LOD: Limit of detection, LLOQ: Lower limit of quantification, HIV: Human immunodeficiency virus, HBV: Hepatitis B virus, HCV: Hepatitis C virus. * LODs for HIV-1 Group O and HIV-2 are not shown here

RESULTS

DISCUSSION

The data presented in this study demonstrate the ability of NAT applied in minipools to detect viral infections that are missed by serology. The data on viral load quantification and follow-up testing further indicate the potential high risk of transmission through the blood donations declared serononreactive, that were averted by NAT testing. Our study showed a high seroreactivity of 1.45% among all donors with HCV. Studies in the past have reported HCV seroprevalence among blood donors in Punjab to be around 1.38%,^[21] and 1.45%.^[22] As per the NACO/National Blood Transfusion Council (NBTC) report in 2016, the anti-HCV reactivity among donors in the blood center in Punjab was 1.35% in 2016.^[11] This high seroreactivity for HCV in Punjab, where our blood center is located, is a reflection of the high seroprevalence of HCV (3.2%) in the general population of Punjab.^[13] The higher seroreactivity observed for HCV in our study could be due to the use of a highly sensitive serological assay using the advanced ECLIA technology. Interestingly, the second highest seroreactivity in our study was observed for syphilis at 0.80%. Although blood center in Punjab has a high seroreactivity of 0.49% for syphilis,^[11] the rate observed in our study is higher which could be again due to the use of a highly sensitive chemiluminescence based assay as against the rapid plasma reagin (RPR) test commonly employed in blood banks in the state. The seroreactivity for HBV and HIV was similar to other blood banks in the state.^[11]

During the study, NAT in minipools was able to detect 45 HBV and 37 HCV infections from the donations which were “sero-non-reactive.” The NAT yields were thus 1:3337 for HBV, 1:4059 for HCV, and 1:1831 overall, as shown in Table 3.

The combined NAT yields reported from Indian tertiary care settings and stand-alone blood center are quite varied, and range between 1:476 and 1:4403 as per a 2017 review of 11 Indian studies.^[23] A few studies have reported higher NAT yields of 1:354^[24] and 1:974;^[25] both the investigators used enzyme-linked immunosorbent assay (ELISA)-based serological testing. Chandra et al. also demonstrated a decrease in NAT yield by employing chemiluminescence on donations previously tested using ELISA.^[24] Our study reported a higher overall NAT yield (1:1831) than most recent studies, and this could be due to differing prevalence of TTIs. Thus, a high NAT yield for HBV and a higher than usual HCV NAT yield have pushed the combined NAT yield higher in our study. Of note, the NAT yield in this study is comparable to or higher than other studies despite the fact that many other studies have employed individual donation NAT (ID-NAT), whereas we have used MP-NAT.

A high NAT reactivity was observed in our study over and above a high seroreactivity obtained with an advanced serological technology like ECLIA. This indicates a significant benefit of NAT in helping to minimize the risk of infectious donations entering the blood supply. Since each blood bag may be separated into multiple components for transfusion, each NAT yield has the potential to prevent transmission of TTIs to multiple transfusion recipients.

A noteworthy finding of our study is the result of the viral load quantitation in NAT yield samples. However, the initial plan was to quantify all NAT yields; viral load testing could only be done on 65/82 NAT yields due to inadequate sampling and clotting in a few samples. However, some interesting insights were revealed from this testing. The HCV NAT yields in our study showed very high viral loads ranging from 25.3 to 9.3 × 10⁶. The high HCV viral loads observed in our study are consistent with the rapid doubling time of HCV in early infection. This demonstrates that donations with high viral titers of HCV, having a high potential for transmission, may be missed by serological assays detecting antibodies to HCV, further underlining the importance of narrowing the window period (WP) of detection through detection of the viral genome by NAT. In our study, the viral loads of 19/35 tested HBV NAT yields were below 20 IU/mL. These HBV infections with low viral loads that were missed by serology but picked up by NAT could be either OBI, recent HBV infections at a stage before the appearance of detectable levels of HBsAg, or resolving HBV infection at a stage where HBsAg has declined to below detectable levels but circulating HBV DNA is not yet cleared. There was one case each of HBV and HCV where NAT yield was positive, but the viral load was undetectable. One possible reason for this is that the sensitivities of the HBV and HCV quantitative molecular assays are lower than those for the MPX2 blood screening assay. In either of these cases, the viral load could possibly be below the limit of detection of the quantitative assays; however, the impact of sampling effects also cannot be completely ruled out.^[26] The differences between the viral loads of HCV and HBV echo the differences in the biology of the two viruses.

Out of 82, 11 NAT reactive donors reported back after follow-up and were found to be positive for serology. According to NBTC guidelines,^[27] all initial seroreactive and NAT positive donors should be recalled, given post donation counseling and repeat sample is to be taken for testing. 11 NAT initial reactive donors developed seropositivity (four donors with HBV and seven donors with HCV) and were counseled. The eventual appearance of HBsAg in the four HBV NAT reactive donors and anti-HCV in the seven HCV NAT reactive donors suggests that these were serological WP donations. In our study, all 11 donors with NAT-positive donations seroconverted, thereby affirming the value of NAT in averting certain TTI transmission. A similar finding was reported by Mishra et al. in 2017, where out of 51 seronegative samples showing NAT yield, seropositivity was observed within 6 months – 1 year for 38 donors (31/44 HBV, 2/2 HCV, and 5/5 HIV seroconversions).^[28] In India, where the HBV endemicity is high, the frequency of OBI is often higher than acute infection. In 2014, Doda et al. evaluated the HBV antibody profiles of HBsAg negative, HBV DNA positive donations; out of 18 samples profiled, 12 showed antibodies against HBc (HBV core antigen), signifying OBI, whereas the remaining 6 had no detectable serological markers, indicating that these donors were in the WP of HBV infection.^[29] The HCV NAT yields are majorly expected to be from the serological WP for HCV infection. While occult Hepatitis C infection is also described in the literature, the information about this entity is rather limited, and much research is needed in this area to understand the clinical implications of this condition.^[30] A complete follow-up of all NAT yield donations would be a step toward a more comprehensive understanding of the stage of infection, the clinical implications of such donations, and mitigation of the risk.

Before the implementation of MP-NAT PCR in our set-up, we had been performing ID-NAT using the transcription-mediated amplification-based Procleix Ultrio Plus® (Novartis Diagnostic, USA) on the Procleix Panther system. The purpose of the current study was also to evaluate the overall ease of implementation of the fully automated NAT-PCR system. The cobas MPX2test is a multiplex PCR test that has the ability to simultaneously detect a reactive donation and identify the target virus (HBV, HCV, or HIV) with specific dyes in different channels. This eliminates the need for discriminatory testing in reactive donation samples for separately identifying the target virus and helps the blood bank plan further actions with certainty. Difficulty in confirming reactive donations obtained through IDNAT on the Ultrio Plus assay has been reported, including recently in India, where only 22%^[31] and 41%^[32] of initially reactive samples could be confirmed by repeat testing and viral target discrimination. In our previously published study of the ID-NAT assay, too, only about 83% of the initially reactive results were confirmed.^[15] The algorithm that we used previously for the ID-NAT assay is given in Figure 2.^[15] This discrepancy and non-confirmation lead to uncertainty on the cause of initial reactivity (non-specific amplification, sample contamination, low viral load, etc.) and the viral target, leading to subsequent difficulties in donor recall and counseling and unnecessary deferrals and repeat tests.^[18] In comparison, we found the screening algorithm of the MPNAT PCR assay to be simpler. This algorithm uses NAT for testing only the sero-nonreactive donations; this reduces the number of tests to be carried out on reactive donor samples. Further, since the screening is done in minipools of multiple donations (in our case, six donations), there are additional benefits of fewer number of tests, higher specificity, and lower overall cost. Moreover, PCR is a time-tested technology. A few additional benefits that we found as a user of the MPNAT technology are minimal maintenance and hands-on time (since the system is completely automated), availability of ready-to-use reagents, no requirement of test calibrations, provision of internal control for monitoring test performance in each individual test, and incorporation of the AmpErase enzyme to reduce potential contamination by previously amplified material.

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Figure 2:

Screening algorithm for individual donation-nucleic acid testing used by our blood bank previously. ID-NAT: Individual nucleic testing, dHIV: Discriminatory Human Immuno Deficiency Virus, dHCV: Discriminatory Hepatitis C, dHBV: Discriminatory Hepatitis B

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The data generated by this study and their interpretation are reinforced by the size of the study, the additional quantitation data available, and follow-up serology. This large study demonstrated the utility of the highly sensitive cobas Taq Screen MPX2 to screen pooled samples and obtain high NAT yields. It also identified the viral loads and stage of infection of these donations, which can be used to derive the risk to blood safety and public and individual health. The investigation also illustrated the high convenience and ease of using automation and minipool testing. However, there are some limitations in the present study. The use of additional serological markers like antibody against Hepatitis C core antigen (HBc) in follow-up testing would have helped characterize OBIs. The use of a higher sensitivity quantitative molecular assay would have enabled us to demonstrate that the blood screening assay detects samples with much lower viral loads as has been shown by other authors.^[24] Finally, the cost-effectiveness of the incorporation of MPNAT, in addition to serological testing and comparison to ID-NAT, was not assessed; further studies are required in this direction.

CONCLUSIONS

The present study has reconfirmed the high burden of TTIs in blood donations in India despite mandatory serological screening and has indicated that the routine application of MP-NAT screening in Indian blood centers would be a move one step closer to safer blood. Cardinal features required for adoption and smooth running in blood centers of varied sizes and donor infection prevalence include reliability, ensured by the use of a highly sensitive assay, ease of operation, ensured by the use of a fully automated system, and an uncomplicated testing algorithm. MP-NAT performed using the cobas MPX2 suitably fulfills both these conditions.