ID-NAT (Individual donor nucleic acid testing)- MP-NAT (mini-pool testing)- window period Reverse transcription-polymerase chain reaction (RT PCR) Transcription mediated amplification (TMA) automated triplex NAT assays RT- PCR- Steps TMA steps RT PCR vs TMA difference NAT for HIV Hepatitis B hepatitis C

1. NAT Testing-
Current Screening
Presenter- Dr. Shiny
Moderator- Dr. Nidhi Bansal

2. Introduction
•Nucleic acid testing (NAT), also termed nucleic acid amplification technology
(NAAT), is a molecular amplification technology that targets, amplifies, and
detects the genetic material (RNA/DNA) of a pathogen (virus, bacteria, or
parasite).
•highly sensitive and specific for viral nucleic acids.

3. •NAT reduces the window period of HIV, HBV, and HCV infections by early
detection of the viral genome.
•Main objective of NAT testing- to reduce the risk of transmission of the virus to
recipients by detecting the minimum amount of viral copies of all known
genotypes and subgroups.

4. Types
Two kinds of testing strategies used worldwide are-
•ID-NAT (Individual donor nucleic acid testing)- a blood sample from a single
donor is tested individually
•MP-NAT (mini-pool testing)- samples from different donors are pooled together
in a range of 4, 6, 8, 16, 18, 20 to 96 and tested as one sample

5. Comparison
•ID-NAT screening will only slightly increase the detection of infected donors but
significantly increase the testing cost as compared to MP-NAT.
• There are chances of false-positive results with ID-NAT due to higher sensitivity
•There are chances of missed positive donors in MP-NAT due to lower sensitivity.

6. Difference from serology:
The most sensitive NAT (in individual format) shortens this window period.
•NAT also benefits by resolving false reactive donations of the serological
methods, which is very important for the donor notification and counselling.
•But NAT testing is not an alternative to serological testing because long term
chronic TTIs have sufficient amount of antibodies with a low viral load which
can be detected only on serological testing.
ORIGINAL WINDOW
PERIOD
SHORTENED
WINDOW PERIOD
HIV 14-28 days 2.9 days
HBV 42-55 days 10.3 days
HCV 50-80 days 1.3 days

7. NAT testing methods for screening of blood donors:
For blood screening, current in-house and commercial assays are extensively
based on two techniques:
 Reverse transcription-polymerase chain reaction (RT PCR)
 Transcription mediated amplification (TMA)

8. CURRENT SCREENING
In India and around the world, automated triplex NAT assays for screening blood for
HBV, HCV and HIV are mainly available from two manufacturers –
Gen-Probe-Novartis (GPN)- based on Transcription Mediated Amplification (TMA)
Roche Molecular Systems (RMS)- based on RT-PCR
GPN RMS
ProcleixUltrio cobasTaqScreen MPX
ProcleixUltrio Plus cobasTaqScreen MPX v2.0
ProcleixUltrio Elite

9. Reverse transcription PCR (RT PCR)
• uses a Reverse Transcriptase enzyme that converts the target RNA into
complementary DNA (cDNA).
•This cDNA is then amplified like in a standard PCR which involves 3 basic steps
1. target denaturation
2. annealing of primers
3. extension of the new strand

10. RT- PCR- Steps

12. Step 1- Specimen preparation
Lysis reagent carries out viral lysis leading to release of nucleic acids.
Due to net negative charge of nucleic acid in presence of lysis reagent, the
nucleic acids bind to added magnetic particles
unbound material is washed out
Purified nucleic acids are eluted

13. Step 2- Amplification
Reverse transcription and amplification are carried out by a recombinant enzyme Z05 DNA polymerase.
Z05 DNA polymerase has reverse transcriptase and DNA polymerase activities in presence of manganese.
Reverse transcriptase activity generates cDNA in case of HCV and HIV.
After reverse transcription amplification of cDNA is carried out.
During PCR amplification, the thermal cycling denatures the target amplicon to ssDNA and DNA
polymerase converts these single strands into double-stranded DNA amplicons.
This process is repeated for multiple cycles, with each cycle number of DNA amplicons get doubled.

14. Step 3- Detection
Detection occurs simultaneously with amplification and sequence-specific dual labelled
probes are used to detect the presence of the target.
These probes are labelled with reporter dye at 5’end and quencher dye at 3’end.
The reporter dye fluorescence is supressed by the quencher dye.
This quenching effect continues till the both dyes are in close proximity and is known as
Forster resonance energy transfer quenching mechanism.

15. During amplification, the probes hybridize to target complementary DNA sequences
and are cleaved by the 5’ to 3’ exonuclease activity of Z05 DNA polymerase at the
time of primer extension.
This leads to breakage of the close proximity of these dyes and fluorescence occurs.
With each PCR cycle a number of probes are cleaved and therefore with each cycle
fluorescence signal increases.

17. Advantages:
•simplicity, flexibility, inexpensive and can amplify damaged DNA.
Disadvantages-
•contamination risk, primer complexity, which can amplify rare species and multi-
temperature requirements

18. Transcription mediated amplification (TMA):
•TMA technique is based on two enzymes
a. RNA Polymerase
b. Reverse Transcriptase
•The Viral RNA target is reverse transcribed into cDNA
•DNA formation is then followed by synthesis of RNA amplicons from DNA by
RNA polymerase.
•The final product of a TMA reaction is RNA amplicons

19. TMA- Steps

21. Step 1- Target capture
The samples are lysed to release viral nucleic acid
Viral genomic sequence specific probes hybridise to the target sequences.
The hybridised nucleic acids are then captured onto magnetic micro particles
Non-specific nucleic acid is washed out to minimize potential inhibitors.

22. Step 2- Amplification
 Carried out with two enzymes- reverse transcriptase and T7 RNA polymerase.
The reverse transcriptase generates cDNA containing promoter sequences for T7
RNA polymerase from the target sequence.
RNA polymerase produces RNA amplicons from cDNA template through
transcription.
It is claimed that billions of copies are generated in less than one hour.

23. Step 3- Detection
Detection is achieved by hybridization protection assay (HPA).
In HPA, sequence-specific single stranded nucleic acid probes labelled with
acridinium ester (AE) hybridise to the RNA amplicons generated through TMA.
The longer-lasting chemiluminescent signal generated by the hybridised probe is
detected by a luminometer and reported in terms of relative light units (RLU).

25. Transcription-mediated amplification- advantages :
1. extraction and amplification takes place in the same tube under isothermal
conditions.
2. The test lends itself to a triplex configuration- HIV, HCV and HBV can be
measured simultaneously.

26. •As RNA can be easily destroyed in a lab setup, the risk of cross-contamination is very low
in a TMA reaction compared to PCR.
•It is an isothermal technique that can occur in a single tube in multiplex format and capable
of detecting many pathogens in one reaction.
•TMA based assays are considered more sensitive compared to conventional PCR assays.
TMA can produce 100-1000 copies of RNA amplicons in one cycle whereas, PCR only
doubles with every cycle. This ends up in billions of amplified products in a shorter time.

27. Internal Control (IC)
Internal control is added to each specimen, control (negative & positive) and assay
calibrator to monitor specimen preparation, amplification and detection procedures. IC is a
target sequence (not related to HBV, HCV or HIV) and a calculated amount of it is added
into the specimen
In Procleix assays IC is detected using a probe with rapid emission of light.
In case of cobas MPX assays different fluorescent dyes are used for IC which are detected
at different wavelengths

31. NAT for HCV
•HCV NAT reduces the window period from 60-80 days to a minimum of 1.3 days.
•During the window phase, the high viral titres of HCV viral doubling time of <1 day,
made HCV the best candidate for the application of NAT to identify the highest
yield of contaminated donations, even when samples were pooled.

32. NAT for HIV
In contrast to HCV, the window period for HIV is much shorter and the viral
doubling rate, as well as viral load, is much lower during the window phase.
HIV NAT reduces the window period from 14-28 days to a minimum of 2.9 days.

33. NAT for HBV
•For HBV, the situation is slightly difficult.
•The replication rate of HBV is very low, with a doubling time of 2.6 days;
therefore, HBV NAT reduces the window period from 42-55 days to a minimum
of 10.3 days.

34. •The nation-wide factors to implement NAT is for-
(i) Prevalence of TTI viruses in the donor population
(ii) voluntary or replacement donors
(iii) Lookback programme for donor monitoring
(iv) Implementation of the haemovigilance programme in the country
(v) Cost v/s benefit comparisons between NAT testing and disease treatment cost.
•The blood centre-level factors are-
(i) Different algorithms and testing assays for serological screening of blood donors
(ii) to detect Regional variations in TTI prevalence.

36. Literature review

37. •First multicenteric study was done by Makroo et al. in 2008 where a total of
12,224 samples along with their serological results were obtained from eight
blood banks in India and were tested individually manually for HIV 1, HCV and
HBV.
•In another study conducted in north India, 18,354 donors were tested by both ID-
NAT and fourth generation enzyme ELISA, 7 were found to be NAT-positive but
ELISA-negative.

38. •Till date, 58/2550 (2%) blood banks of India are doing NAT testing but all of
them have not published their results.
•Majority of the centers have used ID-NAT (Individual NAT) protocol and 21
blood banks are using minipool format of the test.
•For individual infection, NAT yield from the pooled data showed HIV in
1:66,000, Hep C virus 1:5484 and Hep B in 1:1761 seronegative donors.

39. The article by Chatterjee et al. has compared the sensitivity of ID- and MP-NAT.
The authors observed that samples with high viral load were detected by all
dilutions, but 67% of samples of low viral load are missed by MP-NAT and
concluded that ID-NAT is ideal methodology for TTI screening.

40. Major barriers in implementing routine NAT testing in India is its high cost and
lack of technical expertise in most of the blood centers.
In United Kingdom, NAT has reduced the risk of HCV by 95% and that of HIV
by 10%.
The American Red Cross implemented automated triplex NAT for HIV, HCV
and HBV in June 2009.

43. REFERENCES
• Hans, R., & Marwaha, N. (2014). Nucleic acid testing-benefits and constraints. Asian journal of
transfusion science, 8(1), 2–3. https://doi.org/10.4103/0973-6247.126679
•Rajput M. K. (2016). Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood
Screening in India. Journal of clinical and diagnostic research : JCDR, 10(2), KE01–KE4.
https://doi.org/10.7860/JCDR/2016/16981.7319
•DGHS. Tranfusion Medicine Technical Manual 3rd edition