|Year : 2020 | Volume
| Issue : 1 | Page : 50-52
Nucleic acid amplification testing – A paradigm shift
Iruvaram Sudhir Chaitanya Kumar, B Suresh Babu, KV Sreedhar Babu
Department of Transfusion Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
|Date of Submission||20-Aug-2019|
|Date of Acceptance||24-Sep-2019|
|Date of Web Publication||2-Jun-2020|
Iruvaram Sudhir Chaitanya Kumar
Associate Professor, Sri Venkateswara Institute of Medical Sciences, Tirupati 517 507, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Chaitanya Kumar IS, Babu B S, Sreedhar Babu K V. Nucleic acid amplification testing – A paradigm shift. J Clin Sci Res 2020;9:50-2
|How to cite this URL:|
Chaitanya Kumar IS, Babu B S, Sreedhar Babu K V. Nucleic acid amplification testing – A paradigm shift. J Clin Sci Res [serial online] 2020 [cited 2020 Aug 15];9:50-2. Available from: http://www.jcsr.co.in/text.asp?2020/9/1/50/285718
A large number of blood transfusions happen daily around the world as a life-saving procedure for road traffic accidents victims, severe anaemia patients, burns patients, etc., However, blood transfusion is an irreversible event that can never be claimed as safe due to the possibility of transfusion transmissible infections and various transfusion reactions despite the use of state-of-the-art technologies. Conventionally, in developing countries, blood and its components are screened for serological markers for various transfusion transmissible infections endemic in the region. Nucleic acid testing (NAT) of donors was implemented to reduce the seronegative window periods of donors for infectious markers of human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) from 21 days, 38 days and 60 days to 2.93 days, 10.34 days and 1.34 days, respectively, with the implementation of NAT in addition to serological screening methods such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay screening.
At present, around 33 countries in the world have implemented NAT for HIV and around 27 countries for HBV, similarly for HCV in 23 countries NAT is mandatory. In India, serological testing of all blood donations is mandatory for anti-HIV 1/2, hepatitis B surface antigen (HBsAg), anti-HCV, syphilis and malaria, while NAT is not mandatory. Although NAT has a shorter window period, NAT testing is not a replacement to conventional serological testing because of considerable sero-yield. Sero-yield may be used to describe seropositive and NAT-negative samples and can be found in cases of HIV-, HBV- or HCV-positive patients. Similarly, NAT yield is term used to describe seronegative units found positive for nucleic acids of HIV, HBV or HCV. The likelihood or residual risk is the chance that an individual is in window period and be missed by the techniques used for screening thereby transmitting the infection. It can be calculated by multiplying the window period with incidence of disease in the general population. Newer generation of screening tests (like NAT) reduces the window period thereby reducing the residual risk of disease transmission to patients.
NAT is the current most sensitive test in screening blood donors. The principle is based on polymerase chain reaction (PCR) in which amplification of specific strands of nucleic acid either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) pertaining to the virus to be detected using either polymerase enzymes (DNA polymerase for DNA viruses or reverse transcriptase followed by DNA polymerase for RNA viruses) finally yielding multiple copies specific segments of DNA or using enzymes such as reverse transcriptase (transcription-mediated assay) followed by RNAse (nucleic acid sequence-based amplification) and a T7 polymerase finally yielding many copies of RNA. Finally, from a single copy of virus, a large number of copies of a specific viral nucleic acid sequence are amplified with multiple cycles of annealing and extension utilising included primers and nucleotides. The final amplicon (viral sequence amplified) is detected using various probes in indicating various infections.
Currently, only two Food and Drug Administration-approved automated systems are available for market use with principles of real-time PCR and transcription-mediated amplification.
The global scenario of donor screening, scenario in developed and developing countries is shown in [Table 1]. Infectious disease testing is mandatory in most countries of the world with certain limits subject to their socioeconomic and epidemiological backgrounds.
|Table 1: Global pattern of transfusion transmissible infectious disease screening|
Click here to view
A pilot study was performed in our institute over a period of 1 month at a tertiary care teaching referral institute (April 2019) to test all the blood donations concurrently with conventional ELISA and the modern NAT to find out any differences in the pattern of recognition of viral markers for HIV, HBV and HCV and the possible risk of transmission of transfusion-transmissible infections such as HIV, HBV and HCV.
During the study, a total of 1075 donors donated blood voluntarily and out of which 34 units were issued untested to a local government hospital for further testing and processing as a part of the government procedure. The rest 1041 donors were tested for anti-HIV 1 and 2 antibodies, p24 antigen by ErbaLisa HIV Gen4 (Transasia Biomedical Limited, Daman), HBsAg by Merilisa HBsAg (Meril Diagnostics, Vapi), anti-HCV antibodies by ErbaLisa HCV Gen3 (v2) (Transasia Biomedical Limited, Daman) and nucleic acids of HIV, HBV, HCV by Roche TaqScreen, MPX V2.0 (Roche Diagnostics, Switzerland) concurrently on plasma samples in minipools prepared using Hamilton Microlab Star IVD system (Hamilton Company, US). If any minipool is reactive, then the secondary run of individual samples was performed for the detection of viral nucleic acids. All biosafety procedures and biomedical waste protocols currently available were followed.
A total of 13 samples were found to be reactive for HBV DNA and 1 sample for HIV RNA by NAT in comparison to only 12 HBsAg-reactive samples and 1 sample reactive for anti-HIV 1 and 2 antibodies, p24 antigen by ELISA.
To summarise, a sample was found nonreactive in conventional ELISA testing for HBsAg and was found to be repeat reactive for HBV DNA in both minipool testing and individual secondary run. Hence, the NAT yield was calculated as 1 in 1041 for HBV and zero in 1041 for HIV and HCV during a single month of comparative study. This unit was utilised to prepare two components namely packed red cells and fresh frozen plasma, respectively. Early detection of this unit by NAT in contrast to ELISA has led to saving of two lives from possible transfusion-transmitted hepatitis B infection and related comorbidities.
Similar results were obtained in a meta-analytical study from India, which compiled that a total of 3,89,387 blood units have been NAT tested from various parts of the country in 11 studies and a personal communication with a NAT yield varying from 1 in 476 to 1 in 4403. For individual infection, NAT yield from the pooled data showed HIV in 1:66,000, HCV 1:5484 and hepatitis B in 1:1761 seronegative donors in the above study.
To conclude, NAT testing has become a second layer of safety in addition to the present serological methods in most developed countries and developing countries, although not able to replace the serological methods of testing. The NAT yield can definitely protect a greater number of potential recipients and reduce the residual risk of transfusion transmissible infection among the recipients.
We would like to thank the staff of the Department of Transfusion Medicine for their prompt support in sampling and processing requests.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Screening Donated Blood for Transfusion Transmissible Infections, Recommendations. Geneva, Switzerland: World Health Organization; 2010.
Manisha S, Shweta M. Nucleic Acid Amplification testing (NAT): An innovative Diagnostic approach for enhancing blood safety. Natl J Lab Med 2017;6:1-6.
Roth WK, Busch MP, Schuller A, Ismay S, Cheng A, Seed CR, et al
. International survey on NAT testing of blood donations: Expanding implementation and yield from 1999 to 2009. Vox Sang 2012;102:82-90.
Marwaha N, Sachdev S. Current testing strategies for hepatitis C virus infection in blood donors and the way forward. World J Gastroenterol 2014;20:2948-54.
Mark KF. AABB Technical Manual. 19th
ed. Maryland, USA: AABB Press. 2017.
Ghosh K, Mishra K. Nucleic acid amplification testing in Indian blood banks: A review with perspectives. Indian J Pathol Microbiol 2017;60:313-8.
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