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Review Article IMPACT OF ROUTINE INDIVIDUAL BLOOD DONOR NUCLEIC ACID TESTING (ID NAT) FOR HIV-1, HCV AND HBV ON BLOOD SAFETY IN A TERTIARY CARE HOSPITAL R.N.Makroo Director, Department of Transfusion Medicine, Indraprastha Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India. Background: India has a high prevalence of HIV, HCV and HBV among general population and blood donors. We implemented routine Nucleic Acid Testing (NAT) apart from regular serological testing to strengthen our blood safety programme. Objectives: (i) to observe NAT yield for these viruses among our hospital’s blood donors. (ii) To improve safe blood transfusion practices. Materials and Methods: 22277 samples were individually tested by serology for HIVAb, HCVAb, HBsAg, HBcAb and by NAT- Procleix® Ultrio® Assay (Chiron Corp. Emeryville, CA) for simultaneous detection of HIV-1, HCV, and HBV. Results: There were 7 NAT yield cases (3 HCV and 4 HBV) which were reactive for NAT but serologically non reactive by mandatory serological tests. Interpretation & Conclusions: Our observed NAT yield for all 3 viruses is 1 in 3182 (0.03%), which is very high when compared to other countries. NAT can reduce the Transfusion Transmitted Infections considerably & improve blood safety. Key Words: Nucleic Acid Testing (NAT), Blood, HIV-1, HCV, and HBV. INTRODUCTION PROTECTION of blood supply from virus-infected donation has been at a very high level due to effective donor selection and testing with the latest techniques. Blood safety, especially in respect of viral infections like HIV, HBV and HCV, has improved considerably in the developed world because of dependence on voluntary, non-remunerated regular blood donors, latest serological testing for infectious markers and incorporation of Nucleic Acid Testing to narrow the window period in blood donations. While no blood transfusion is absolutely safe, countries with well-managed blood transfusion services like Japan, USA and Western Europe have achieved commendable levels of blood safety yet make no claims of providing 100% safe or zero risk blood. The blood safety status in India is still under lot of questions due to the recent reported cases of Transfusion Transmitted Infections from some of our premier hospitals. Currently the seroprevalence of anti-HIV-1, anti-HCV, and HBsAg in Indian blood donors is 0.5%, 0.4%, and 1.4% [1], respectively compared to 0.0097%, 0.3%, and 0.07% for the 3 viruses in US blood donors [2]. Improving blood safety in India presents unique challenges. In our country, blood collection, testing and distribution are carried out by 2212 licensed blood banks that are operated both privately and by the government. Despite the current practice of screening blood with government approved kits, chiefly employing enzyme immunoassay technologies, a recent survey of blood transfusion practices noted that testing for potential transfusion-transmitted infections is unsatisfactory and poorly regulated in most blood banks, regardless of their type and location. India is the largest country of this region with a population of more than 1.2 billion, which includes about 2.5 million HIV (NACO), 43 million HBV [3] and 15 million HCV [4] infected persons. Blood Transfusion Services (BTS) in India are mainly hospital based and is governed by Drugs & Cosmetic Act. There are 2212 licensed blood banks under different administrative controls i.e. Government 41%, Voluntary 12%, Private hospital 22% & Private Commercial 25 %. There is wide gap in demand & supply of blood as the annual collection is about 6 million against the demand of approximately 9 million. Blood collection from the voluntary blood donors constitutes only 50%. Majority of voluntary donors are first time donors, as there is little concept of regular repeat voluntary blood donation. Each and every unit of blood is screened for HBsAg, anti HIV, anti HCV, malaria & syphilis as per Drugs & Cosmetic rules before issue to patients. Though paid blood donation is illegal in India there is no strong monitoring or expert donor counseling in many blood banks. Most of the blood banks use serological screening methods for the infectious marker screening. Majority of the blood 9 Apollo Medicine, Vol. 4, No. 1, March 2007

Impact of Routine Individual Blood Donor Nucleic Acid Testing (ID NAT) for Hiv-1, Hcv and Hbv on Blood Safety in a Tertiary Care Hospital

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Page 1: Impact of Routine Individual Blood Donor Nucleic Acid Testing (ID NAT) for Hiv-1, Hcv and Hbv on Blood Safety in a Tertiary Care Hospital

Review Article

IMPACT OF ROUTINE INDIVIDUAL BLOOD DONOR NUCLEIC ACID TESTING (ID NAT)FOR HIV-1, HCV AND HBV ON BLOOD SAFETY IN A TERTIARY CARE HOSPITAL

R.N.MakrooDirector, Department of Transfusion Medicine, Indraprastha Apollo Hospitals,

Sarita Vihar, New Delhi 110 076, India.

Background: India has a high prevalence of HIV, HCV and HBV among general population and blood donors.We implemented routine Nucleic Acid Testing (NAT) apart from regular serological testing to strengthen ourblood safety programme. Objectives: (i) to observe NAT yield for these viruses among our hospital’s blooddonors. (ii) To improve safe blood transfusion practices. Materials and Methods: 22277 samples wereindividually tested by serology for HIVAb, HCVAb, HBsAg, HBcAb and by NAT- Procleix® Ultrio® Assay(Chiron Corp. Emeryville, CA) for simultaneous detection of HIV-1, HCV, and HBV. Results: There were 7 NATyield cases (3 HCV and 4 HBV) which were reactive for NAT but serologically non reactive by mandatoryserological tests. Interpretation & Conclusions: Our observed NAT yield for all 3 viruses is 1 in 3182 (0.03%),which is very high when compared to other countries. NAT can reduce the Transfusion Transmitted Infectionsconsiderably & improve blood safety.

Key Words: Nucleic Acid Testing (NAT), Blood, HIV-1, HCV, and HBV.

INTRODUCTION

PROTECTION of blood supply from virus-infected donationhas been at a very high level due to effective donorselection and testing with the latest techniques. Bloodsafety, especially in respect of viral infections like HIV,HBV and HCV, has improved considerably in thedeveloped world because of dependence on voluntary,non-remunerated regular blood donors, latest serologicaltesting for infectious markers and incorporation ofNucleic Acid Testing to narrow the window period inblood donations. While no blood transfusion isabsolutely safe, countries with well-managed bloodtransfusion services like Japan, USA and Western Europehave achieved commendable levels of blood safety yetmake no claims of providing 100% safe or zero riskblood. The blood safety status in India is still under lot ofquestions due to the recent reported cases of TransfusionTransmitted Infections from some of our premierhospitals. Currently the seroprevalence of anti-HIV-1,anti-HCV, and HBsAg in Indian blood donors is 0.5%,0.4%, and 1.4% [1], respectively compared to 0.0097%,0.3%, and 0.07% for the 3 viruses in US blood donors [2].Improving blood safety in India presents uniquechallenges. In our country, blood collection, testing anddistribution are carried out by 2212 licensed blood banksthat are operated both privately and by the government.Despite the current practice of screening blood withgovernment approved kits, chiefly employing enzyme

immunoassay technologies, a recent survey of bloodtransfusion practices noted that testing for potentialtransfusion-transmitted infections is unsatisfactory andpoorly regulated in most blood banks, regardless of theirtype and location.

India is the largest country of this region with apopulation of more than 1.2 billion, which includesabout 2.5 million HIV (NACO), 43 million HBV [3] and15 million HCV [4] infected persons. BloodTransfusion Services (BTS) in India are mainly hospitalbased and is governed by Drugs & Cosmetic Act. Thereare 2212 licensed blood banks under differentadministrative controls i.e. Government 41%,Voluntary 12%, Private hospital 22% & PrivateCommercial 25 %. There is wide gap in demand &supply of blood as the annual collection is about 6million against the demand of approximately 9 million.Blood collection from the voluntary blood donorsconstitutes only 50%. Majority of voluntary donors arefirst time donors, as there is little concept of regularrepeat voluntary blood donation. Each and every unit ofblood is screened for HBsAg, anti HIV, anti HCV,malaria & syphilis as per Drugs & Cosmetic rules beforeissue to patients. Though paid blood donation is illegalin India there is no strong monitoring or expert donorcounseling in many blood banks. Most of the bloodbanks use serological screening methods for theinfectious marker screening. Majority of the blood

9 Apollo Medicine, Vol. 4, No. 1, March 2007

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Apollo Medicine, Vol. 4, No. 1, March 2007 10

Review Article

collected is used as whole blood as blood componentpreparation is just 15-20%. Increased voluntarydonation, availability of safe blood, componentpreparation and optimum utilization of blood productsare the key needs here.

• Nucleic Acid Testing (NAT) is a method of testingblood that is more sensitive than conventional teststhat require the presence of antibodies to trigger apositive test result.

• NAT works by detecting the low levels of viralgenetic material present when an infection occursbut before the body begins producing antibodies inresponse to a virus.

• NAT significantly reduces the ‘window period’ orthe time between donor exposure to the virus and theappearance of antibodies. By decreasing the windowperiod, it allows for earlier detection of the infectionand thus further decreases the possibility oftransmission via transfusion.

• NAT may also detect mutants, occult cases and falsenegatives from serology.

• When combined with Individual Donor Testing(IDT) – it provides the most sensitive and specificscreening platform for blood screening.

• As a screening tool, individual donor NAT detectsinfection before serological tests -10-16 days earlierfor HIV-1, 49-65 days for HCV, and 25-36 days forHBV [5,6].

MATERIALS AND METHODS

From 1st April 2006 to 30th June 2007, we tested22277-blood donor’s samples by Nucleic Acid Test(NAT) apart from routine serological screening for HIVantibody, HCV antibody; and Hepatitis B surface antigen(HBsAg) and Hepatitis B core antibody.

NAT was conducted at the Department ofTransfusion Medicine, Indraprastha Apollo Hospital,New Delhi using the Procleix® Ultrio® Assay (ChironCorporation, Emeryville, CA) according tomanufacturer’s instructions. All samples were testedindividually. The Procleix® Ultrio® Assay is a multiplextest which provides simultaneous detection of HIV-1RNA, HCV RNA and HBV DNA in human plasma.Using Transcription Mediated AmplificationTechnology (TMA).

The TMA Assay involves three main steps utilizingthree proprietary technologies: (i) target capture of viral

nucleic acid, (ii) transcription-mediated amplification,and (iii) hybridization protection assay, all performed ina single tube. All three assays incorporate an internalcontrol to validate each reaction.

Samples found reactive in the Ultrio test were laterretested for HIV-1, HCV and HBV using discriminatoryassays. The Procleix® HIV-1, HCV, and HBVDiscriminatory Assays utilize the same steps as theProcleix Ultrio Assay (target capture, TMA, HPA andDKA) with one difference: HIV-1 specific, HCVspecific, or HBV specific probe reagents are used inplace of the Procleix Ultrio Assay Probe Reagent.

RESULTS

Among the 22277 samples tested total 2907(13.04%) samples were reactive by serology. Amongthese 2907 seroreactive samples 58 (0.26%) werereactive for HIV antibody, 98 (0.44%) for HCVantibody, 291 (1.31%) for HBsAg and 2460 (11.04%) forHBcAb.

Out of these 22277 samples 256 (1.15%) sampleswere reactive for NAT. Among these 256 NAT reactivesamples, 19 (0.085%) samples were reactive for HIV-1,35 (0.16%) were HCV reactive and 202 (0.91%) sampleswere HBV reactive by NAT.

The combined yield (seronegative/NAT reactive) forHIV-1, HCV and HBV was 7 (0.03%). Among these 7NAT yield samples 4 samples were tested reactive forHBV (0.018 %) and 3 were HCV reactive (0.013 %).

Table 1 and 2 depicts the detail of Elisa and NATresults.

DISCUSSION

Among the 22277 samples tested in our hospitalsince the implementation of routine NAT screening, 7 (1/3182) samples were tested NAT reactive which wereserologically negative for any of the three viruses.Among these 7 NAT yield samples 4 (1/5569) were HBVNAT reactive and other 3 (1/7246) samples were HCV

TABLE 1: ELISA Result

Total no of HIVAb HCVAb HBsAg HBcAbsample tested

22277 58 98 291 2460% Reactivity 0.26% 0.44% 1.31% 11.04%

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Review Article

11 Apollo Medicine, Vol. 4, No. 1, March 2007

NAT reactive. The prevalence of the 3 viruses in oursample was 0.08%, 0.16% and 0.91% for HIV-1, HCVand HBV respectively.

Since each unit may be transfused to multiplepatients after component separation, actual number ofinfections transmitted through transfusion would bemuch higher (~21 infected components).

In our earlier multicentric pilot study of 12,224 blooddonor samples tested from 8 different blood banks inIndia, there were 8 NAT yields (1 in 1528) (0.065%; 95%CI 0.028%- 0.129%): 1 in 12,224 (0.008%), for bothHIV-1 mono-infection & HCV-HIV-1 co-infection and 1in 2037 (0.049%) for HBV, samples that were non-reactive by serology and NAT reactive. The difference ofnumber of yields in the pilot study and routine screeningmay be due to the better pre-donation counseling and useof highly sensitive serological screening in our hospital[7].

If we compare our routines NAT results with otherdeveloping countries like Singapore, Thailand, Korea,Hong Kong and South Africa then we could see thatSingapore has a NAT yield of 1 in 24,567 [8] which isabout 7.7 times lower than us, NAT yield in Hong Kongis 1 in 400,000 [9] which is about 126 times lower thenus, Thailand has a NAT yield of 1 in 25000 [9] which isabout 8 times lower than our yield and the routine NATyield in South Africa is 1 in 14485 [10] which is about4.5 times lower than us. One of the reasons for this loweryield is that these countries mostly collect blood samplesthrough voluntary blood donations, whereas in ourhospital almost 90% donations were collected throughreplacement donors.

In India, by using Procleix Ultrio Assay in IDT, wecould interdict an estimated 2,000-3,000 infecteddonations annually that would have been missed if only

serological screening were used. Since each unit may betransfused to multiple patients after componentseparation, actual number of infections transmittedthrough transfusion would be much higher. Between5000-8000 infected components that would have beenmissed if only serological screening were used.

The choice of ID NAT over pool testing alsocontributed in our high yield rate. Since by pooling thesensitivity of NAT decreases we chose ID NAT forscreening of all the samples. Similarly most of the otherNAT user countries of this region are now moving frompooling to IDT. This has also been seen in South Africa,Thailand, Singapore and many European countries.

Blood safety is a special challenge in India because ofthe high prevalence HIV, HCV, and HBV, the relativelylow percentage of volunteer donors, and the lack ofstandardization of screening procedures among themultitude of blood collection centers.

CONCLUSION

In spite of considerable awareness, blood transfusionservices suffer from inadequate political commitment,priority, fragmentation and lack of resources. Whilecurrent serological tests have made a significantdifference, we are nowhere close to world standards.NAT would make our blood supply comparable to thehighest standards in the world. NAT when performed onindividual donor samples provides a highly sensitive,cost effective solution for protecting recipients of bloodtransfusion from HIV, HBV and HCV. Many of thecountries have now decided to add HBV NAT in IDTrather than HBcAb to weed out Window Periodinfections and decrease unnecessary wastage of Bloodand its products. Potential developing countries likeIsrael, Egypt, Brazil, Thailand, Malaysia, and Indonesiahave implemented NAT in phased manner. Thailand has

TABLE 2: NAT Result

Among 22277samples tested HIV HCV HBV Combined Total

NAT Reactive 19 35 202 256(0.085%) (0.16%) (0.91%) (1.15%)

NAT Yield 0 3* 4 ** 7(0%) (0.013%) (0.018%) (0.03%)

Yield Rate 0 1/7246 1/5569 1/3182

* 1 sample is HCV NAT reactive but HCV Ab negative & HBcAb reactive1 sample is HCV NAT reactive - HCVAb negative & HBsAg reactive

** 2 samples are HBV NAT reactive -HBsAg negative but HBcAb reactive

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a model where the Red Cross centralizes NAT testing,and though it is not mandatory, all Bangkok Hospitals &Blood Banks and many from other towns voluntarily getNAT tested by paying for it. This allows BangkokHospitals to provide safer blood to not only its citizensbut also attract Medical Tourism.

It is imperative, that the decision-makers in Indiaunderstand the factors governing the availability of safeblood and take timely and meaningful decisions toprovide safe Blood Transfusion Services for the country.Because of our fragmented blood banking system it willbe necessary to introduce NAT in phased manner in ourcountry. The time has come when serious concerted andcoordinated efforts are required to strengthen the bloodtransfusion services.

REFERENCES

1. Bhatia Rajesh. Blood Transfusion Services in DevelopingCountries of South East Asia. Transfusion Today,December 2005.

2. Dodd RY, Notari EP 4th, Stramer SL. Current prevalenceand incidence of infectious disease markers andestimated window-period risk in the American Red Crossblood donor population. Transfusion 2000; 42 (8): 975-979.

3. T. Kurein et al. Community Prevalence of Hepatitis BInfection and Modes of Transmission in Tamil Nadu, India.IJMR 2005; 121: 670-675.

4. Kaur Paramjit, Basu S. J Postgrad Med 2005; 51: 146 -151.

5. Busch MP, Glynn S, Stramer S, Strong D M, Caglioti S,Wright D, et al. A new strategy for estimating risks oftransfusion-transmitted viral infections based on rates ofinfection of recently infected donors. Transfusion 2005;45:254-264.

6. Comanor L, Holland P. Hepatitis B virus blood screening:unfinished agendas. Vox Sang 2006; 91:1-12.

7. R. N. Makroo et.al. The First Indian Multicenter Evaluationof Individual Donor Nucleic Acid Testing (NAT) forSimultaneous Detection of Human Immuno Deficiencyvirus 1 and hepatitis B and C Viruses (HIV-1, HCV, HBV).Transfusion today. December 2005.

8. D. Teo. 5 years of NAT testing in Singapore. ISBT 2006,Cape Town.

9. Information received from the users in Thailand, HongKong and Korea through personal communication.

10. Heynes A.D.P., Swanevelder J.P., Lelie P.N., CrookesR.L., Busch M.P. The Impact of Individual Donation NATScreening on Blood Safety – The South Africanexperience. ISBT Science Series 2006; 1, 203-208.